Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
小麦族系中染色体构象捕获测序:一个有价值的基因组组装工具   

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Abstract

Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.

Keywords: Tethered conformation capture (构象捕获), Hi-C (Hi-C), Physical mapping (物理图谱), Chromatin interaction (染色质互作), Triticeae (小麦族), Sequencing (测序), Genome assembly (基因组组装), Scaffolding (支架)

Background

Any successful Hi-C experiment should reveal the distance-dependent decay of contact probabilities: the frequency by which two loci juxtapose in three-dimensional space is predominantly determined by their distance in the linear genome (Lieberman-Aiden et al., 2009). This observation motivated the application of Hi-C for physical mapping: the number of Hi-C links between pairs of contigs of a whole-genome shotgun assembly can serve as a proxy of the linear distance between them. This distance information can then be fed into graph algorithms to reconstruct the linear order of scaffolds along the chromosomes (Burton et al., 2013; Kaplan and Dekker, 2013). The three-dimensional proximity information obtained from our TCC approach was employed to order and orient BAC-based super scaffolds in the high-quality barley reference genome (Beier et al., 2017; Mascher et al., 2017). For the Emmer genome assembly, the TCC information was used in a similar manner to validate the scaffolds leading to chromosome-scale assemblies (pseudomolecules) (Avni et al., 2017).

TCC is a modified Hi-C approach in which key reactions (marking of the DNA ends and circularization) are performed on a solid phase rather than in solution (Kalhor et al., 2011). In fact, in human cells the tethering approach yielded improved signal-to-noise ratios, thus leading to a better mapping of low-frequency interactions (Kalhor et al., 2011). This observation prompted us to develop a similar protocol for Triticeae, which is based on the isolation of crosslinked nuclei from young leaves (Hövel et al., 2012) followed by TCC (Kalhor et al., 2011) (Figure 1). Briefly, the chromosome conformation is captured in native leaves by chemical crosslinking using formaldehyde (Hövel et al., 2012), which covalently connects proteins to DNA, as well as proteins to each other. Nuclei are purified, and cysteine residues of proteins contained in the chromatin are biotinylated (Kalhor et al., 2011). The DNA is digested with a restriction enzyme (HindIII) followed by immobilization at low density on streptavidin-coated beads via the crosslinked biotinylated proteins (Kalhor et al., 2011). DNA ends are filled-in, marked with biotin and circularized while tethered to the beads (Kalhor et al., 2011). The crosslinks are reversed, ligation junctions are affinity-purified and provided with adapters for Illumina sequencing to discover genuine pairs of intrachromosomal interaction sites, which were initially captured (Kalhor et al., 2011).

Chromosome conformation capture sequence data is analyzed in the context of genome sequence assemblies. If a chromosome-scale reference genome is available, Hi-C (or TCC) reads can be aligned to it and assigned to restriction fragment predicted in silico from the genome sequence. Subsequently, the number of Hi-C reads linking pairs of restriction pairs can be quantified and the counts be aggregated in larger genome windows (e.g., 1 Mb bins) to obtain estimates of the contact probabilities between pairs of genomic loci (Lieberman-Aiden et al., 2009). These contact probabilities can be used to investigate chromatin organization and its interaction with various biological parameters such as stage of the cell cycle (Naumova et al., 2013) or the epigenomic landscape (Zhou et al., 2013). Chromosome conformation capture sequencing can also be used to reconstruct contiguous, chromosome-scale genomic reference sequences from fragmented sequence assembly. The Hi-C/TCC reads are mapped to a sequence assembly composed of unordered scaffolds, and links between scaffolds are counted and used as a measure of genomic distance. The closer two sequence scaffolds are to each other in the linear genome, the higher the number of Hi-C read pairs linking them. This distance information can then be used to derive a linear order of sequence scaffolds and orient adjacent scaffolds relative to each other (Burton et al., 2013; Kaplan and Dekker, 2013). These approaches have been applied in the sequence assembly of insect (Dudchenko et al., 2017) and Triticeae genomes (Avni et al., 2017; Mascher et al., 2017).


Figure 1. Schematic overview of TCC for Triticeae. Leaves of seven days old plants (I) are harvested for chemical crosslinking of the chromatin (II). The chromatin structure is captured by the formation of covalent bonds between proteins (grey ovals) and between proteins and DNA (line). The orange and light blue segments exemplify two HindIII fragments located on a chromosome. Nuclei are purified (III), and proteins are biotinylated (IV, green pentagon) for subsequent tethering of the complexes to a solid phase. DNA is digested (scissors) using the restriction enzyme HindIII (V) and tethered at low density to streptavidin-coated beads (VI, purple). Ends are marked and filled-in using biotin-14-dCTP (VII, red star). By including dGTPαS in the fill-in reaction, a phosphorothioate bond is introduced. Thereby DNA is guarded against Exonuclease III digestion, which is used at a later step to remove biotinylated nucleotides from non-ligated ends (not shown). Filled-in HindIII sites are ligated ‘on-bead’ (VIII), thereby newly creating NheI restriction sites, which are indicating TCC ligation events. The crosslinking is reversed. DNA is purified (IX) and treated with Exonuclease III (not shown) prior to fragmentation (X). The position of primers (grey triangles) used for controlling the marking and ligation of ends (3C control) is indicated (IX). Ends of the fragmented DNA are repaired and tailed with ‘A’ (not shown). DNA fragments with genuine ligation junctions are affinity-purified based on the incorporated biotin (XI) and provided with Illumina adapters (XII, grey lines) for paired-end sequencing. Ligation products are PCR-amplified (XII; arrowheads: position of primer), size-selected (XIII) and quality controlled (QC). The junctions are revealed by paired-end (PE) sequencing using an Illumina HiSeq2500 device (XIV) followed by bioinformatic analysis (XV). The estimated hands-on time is indicated in days (d). The operating period of the Illumina HiSeq2500 instrument is given for 2x 100 cycles sequencing and depends on the chemistry and type of flowcell (days in brackets). The flowchart is adapted from (Kalhor et al., 2011).

Materials and Reagents

Notes:

  1. We tested all components in independent experiments. However, this list does not imply that alternative products from other manufacturers cannot perform just as well.
  2. The use of the published equipment and chemistry is not indicating any competing interest.

  1. Compost soil (multiple vendors)
  2. Pots (16 cm diameter) (multiple vendors)
  3. Aluminum foil (multiple vendors)
  4. 10 µl Sapphire Low Retention Filtertips (Greiner Bio One International, catalog number: 771265 )
  5. 200 µl Sapphire Low Retention Filtertips (Greiner Bio One International, catalog number: 737265 )
  6. 1,000 µl Sapphire Low Retention Filtertips (Greiner Bio One International, catalog number: 750265 )
  7. 0.2 µm filter, Millex GP (Merck, catalog number: SLGP033RS )
  8. 10 ml and 50 ml syringes (multiple vendors)
  9. 50 ml Falcon tubes (Corning, catalog number: 352070 )
  10. Racks for 50 ml Falcon tubes fitting into the desiccator (multiple vendors)
  11. Disposable 10 ml and 25 ml plastic pipettes (multiple vendors)
  12. Pipette fillers (BRAND, catalog number: 25315 )
  13. Paper towels (multiple vendors)
  14. Appropriate protective gear for working with liquid nitrogen (multiple vendors)
  15. Dewar vessel for transport and storage of liquid nitrogen (multiple vendors)
  16. Miracloth (Merck, catalog number: 475855-1R )
  17. Sefar Nitex 03-55/32 (55 µm mesh opening, 32% open area) (Stefan Kastenmüller GmbH)
  18. Tubes (1.5 ml and 2.0 ml; SARSTEDT, catalog numbers: 72.690.001 and 72.695.500 )
  19. Microscopic slides (76 x 26 x 1 mm) (multiple vendors)
  20. Cover slips (multiple vendors)
  21. Cell counting chamber (Neubauer chamber) and glass cover (Celeromics)
  22. Needle (0.9 x 40 mm, HSW FINE-JECT) (Henke-Sass, Wolf, catalog number: 8300025263 )
  23. Slide-A-LyzerTM Dialysis cassette (0.5-3 ml, cutoff 20 kDa) with float buoys (Thermo Fisher Scientific, catalog number: 66003 )
  24. Parafilm (BRAND, catalog number: 701605 )
  25. Magnetic Particle Concentrator (MPC) for 1.5 ml tubes (DynaMag-2 Magnet) (Thermo Fisher Scientific, catalog number: 12321D )
  26. 15 ml conical tubes (Corning, catalog number: 352196 )
  27. Rubber strap or tape
  28. DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) (Thermo Fisher Scientific, catalog number: 12027 )
  29. PCR-tubes, Sapphire (Greiner Bio One International, catalog number: 652250 )
  30. Snap-cap microTUBEs (with AFA-fiber and pre-split septum) (Covaris, catalog number: 520045 )
  31. Disposable scalpels (multiple vendors)
  32. Polystyrene plugs (self-made) of 0.5 to 1.0 cm thickness fitting snugly into the 50 ml plastic tubes
  33. Seeds (provided by the experimenter)
  34. HEPES sodium salt (4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt) (Carl Roth, catalog number: 7020.2 )
  35. Concentrated HCl (Carl Roth, catalog number: 4625.1 )
  36. Sucrose (Carl Roth, catalog number: 9097.1 )
  37. MgCl2•6H2O (Sigma-Aldrich, catalog number: M2670 )
  38. KCl (Honeywell, Fluka, catalog number: 60130 )
  39. Triton X-100 (Carl Roth, catalog number: 3051.3 )
  40. 100% glycerol (Carl Roth, catalog number: 3783.1 )
  41. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 93482 )
  42. 2-mercaptoethanol (14.3 M) (Carl Roth, catalog number: 4227.1 )
  43. 37% (w/v) formaldehyde solution (containing 10-15% methanol as stabilizer) (Sigma-Aldrich, catalog number: 252549 )
  44. Glycine (Carl Roth, catalog number: 3790.1 )
  45. Liquid nitrogen
  46. Aprotinin (Sigma-Aldrich, catalog number: 10820 )
  47. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  48. Pepstatin (Sigma-Aldrich, catalog number: P5318 )
  49. VECTASHIELD mounting medium with DAPI (VECTOR Laboratories, catalog number: H-1200 )
  50. SDS (Sodium Dodecyl Sulfate) (Serva, catalog number: 20765 )
  51. Tris (hydroxymethyl) aminomethane (Tris base) (Carl Roth, catalog number: 5429.3 )
  52. NaCl (Carl Roth, catalog number: 9265.2 )
  53. EZlink Iodoacetyl-PEG2-Biotin (IPB) (Thermo Fisher Scientific, catalog number: 21334 )
  54. 1,4-Dithiothreitol (DTT) (Carl Roth, catalog number: 6908.1 )
  55. HindIII (100 U/µl) (New England BioLabs, catalog number: R0104T )
  56. NheI (10 U/µl) (Thermo Fisher Scientific, catalog number: ER0971 )
  57. MyOne Streptavidin T1 beads (Thermo Fisher Scientific, catalog number: 65601 )
  58. Na2HPO4 (Carl Roth, catalog number: P030.2 )
  59. KH2PO4 (Carl Roth, catalog number: 3904.1 )
  60. 100 mM dATP (Thermo Fisher Scientific, catalog number: R0142 )
  61. 100 mM dTTP (Thermo Fisher Scientific, catalog number: R0172 )
  62. 2'-Deoxyguanosine-5'-(α-thio)-triphosphate, Sodium salt (dGTPαS), 1:1 Mixture of Rp and Sp isomers (Jena Bioscience, catalog number: NU-424S )
  63. Biotin-14-dCTP (Thermo Fisher Scientific, catalog number: 19518018 )
  64. Klenow DNA polymerase, large fragment (10 U/µl) (Thermo Fisher Scientific, catalog number: EP0052 )
  65. UltraPure BSA (50 mg/ml) (Thermo Fisher Scientific, catalog number: AM2616 )
  66. T4 DNA ligase (5 U/µl) with 10x T4 DNA Ligation buffer and 50% PEG 4000 (Thermo Fisher Scientific, catalog number: EL0011 )
  67. RNase A (DNase-free) (MACHEREY-NAGEL, catalog number: 740505 )
  68. Proteinase K (Thermo Fisher Scientific, catalog number: 25530015 )
  69. Phenol:CHCl3:isoamyl alcohol (25:24:1) (Carl Roth, catalog number: A156.2 )
  70. 8-Hydroxyquinoline (Sigma-Aldrich, catalog number: H6878 )
  71. Isoamyl alcohol (Carl Roth, catalog number: 8930.1 )
  72. CHCl3 (Carl Roth, catalog number: 3313.2 )
  73. Glycogen (Thermo Fisher Scientific, catalog number: R0561 )
  74. 100% ethanol (Carl Roth, catalog number: 9065.4 )
  75. Q5 Hot Start High-Fidelity DNA Polymerase (2 U/µl) and 5x Q5 Hot Start buffer (New England BioLabs, catalog number: M0493S )
  76. Primer for biotin incorporation control (barley):
    Primer 1 (5'- ATCTTCATGCGAGGCAGAGT-3')
    Primer 2 (5'- ACCGTTGAACCATCTTCAGG-3')
    Note: The primers are HPLC purified, 0.2 μmol synthesis scale, dissolved in H2O to 100 µM; contact e.g., Sigma-Aldrich for synthesis.
  77. dNTP Set (100 mM solutions) (Thermo Fisher Scientific, catalog number: R0181 )
  78. dNTP Mix (25 mM each) (Thermo Fisher Scientific, catalog number: R1121 )
  79. MinElute Gel Extraction Kit (QIAGEN, catalog number: 28606 )
  80. Bromophenol Blue (Carl Roth, catalog number: A512.2 )
  81. 10x NEBuffer 1 buffer (New England BioLabs, catalog number: B7001S )
  82. UltraPure Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  83. Exonuclease III (100 U/µl) from E. coli (New England BioLabs, catalog number: M0206S )
  84. AMPure XP beads (Beckman Coulter, catalog number: A63881 )
  85. 10x Tango (Thermo Fisher Scientific, catalog number: BY5 )
  86. 100 mM ATP (Thermo Fisher Scientific, catalog number: R0441 )
  87. T4 DNA polymerase (5 U/µl) (Thermo Fisher Scientific, catalog number: EP0062 )
  88. T4 polynucleotide kinase (10 U/µl) (Thermo Fisher Scientific, catalog number: EK0031 )
  89. Klenow DNA polymerase, large fragment (10U/µl) (Thermo Fisher Scientific, catalog number: EP0052 )
  90. Klenow fragment, 3'→5' exo- (5 U/µl) (New England BioLabs, catalog number: M0212L )
  91. Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific, catalog number: 65001 )
  92. DNA LoBind tubes (1.5 ml) (Eppendorf, catalog number: 0030108051 )
  93. TruSeq DNA Single Indexes Set A (Illumina, catalog number: 20015960 )
  94. Library amplification primer:
    Primer 3 (5'-AATGATACGGCGACCACCGAGAT-3')
    Primer 4 (5'-CAAGCAGAAGACGGCATACGA -3')
    Note: The primers are HPLC purified, 0.2 μmol synthesis scale, dissolved in H2O to 100 µM; contact e.g., Sigma-Aldrich for synthesis.
  95. SYBR-Gold (Thermo Fisher Scientific, catalog number: S11494 )
  96. GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific, catalog number: SM0373 )
  97. Glacial acetic acid (Carl Roth, catalog number: 3738.5 )
  98. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
  99. 100% isopropanol (Carl Roth, catalog number: 6752.4 )
Note: Prepare the following solutions (#100-#107) as described in Green and Sambrook (2012).
  1. 1 M Tris-HCl, pH 8.0 (100 ml)
  2. 1 M Tris-HCl, pH 7.4 (100 ml)
  3. 1 M Tris-HCl, pH 7.5 (100 ml)
  4. 0.5 M EDTA, pH 8.0 (100 ml)
  5. 1 M DTT (20 ml)
  6. Phenol:CHCl3:isoamyl alcohol (25:24:1) with 0.1% 8-Hydroxyquinoline
  7. CHCl3:isoamyl alcohol (24:1)
  8. 1 M MgCl2
  9. 1 M HEPES, pH 8.0 (see Recipes) 
  10. 2 M sucrose (see Recipes)
  11. 1 M KCl (see Recipes)
  12. 10% (v/v) Triton X-100 (see Recipes)
  13. Nuclei Isolation Buffer (NIBF) with formaldehyde (see Recipes)
  14. 2 M glycine (see Recipes)
  15. 1 mg/ml Aprotinin (see Recipes)
  16. 1 mg/ml Leupeptin (see Recipes)
  17. 1 mg/ml Pepstatin (see Recipes)
  18. Nuclei Isolation Buffer with protease inhibitors (NIBP) (see Recipes)
  19. Sucrose cushion (see Recipes)
  20. Nuclei Isolation Buffer with 1.5 M sucrose (NIBS) (see Recipes)
  21. 2% (w/v) SDS (see Recipes)
  22. 4 M NaCl (see Recipes)
  23. Wash buffer 1 (see Recipes)
  24. 25 mM IPB (see Recipes)
  25. 10x NEBuffer 2 (see Recipes)
  26. 1x NEBuffer 2 (see Recipes)
  27. HindIII (10 U/ µl) (see Recipes)
  28. Dialysis buffer (TE buffer) (see Recipes)
  29. Phosphate Buffered Saline with Tween 20, pH 7.4 (PBST) (see Recipes)
  30. 25 mM neutralized IPB (see Recipes)
  31. 10 mM dATP (see Recipes)
  32. 10 mM dTTP (see Recipes)
  33. Wash buffer 2 (see Recipes)
  34. Wash buffer 3 (see Recipes)
  35. 10x ligation buffer TCC (see Recipes)
  36. 10 mg/ml BSA (see Recipes)
  37. Extraction buffer (see Recipes)
  38. 25 mg/ml RNase A (see Recipes)
  39. 20 mg/ml Proteinase K (see Recipes)
  40. 5 mg/ml glycogen (see Recipes)
  41. 80% ethanol (see Recipes)
  42. 70% ethanol (see Recipes)
  43. 3 M sodium acetate, pH 5.2 (see Recipes)
  44. EB (see Recipes)
  45. EBT (see Recipes)
  46. Primer 1 + 2 (10 µM each) (see Recipes)
  47. 10 mM dNTP mix (see Recipes)
  48. 2.5 mM dNTP mix (see Recipes)
  49. TLE (see Recipes)
  50. Tween Wash Buffer (TWB) (see Recipes)
  51. 2x Binding Buffer (2x BB) (see Recipes)
  52. 1x Binding Buffer (1x BB) (see Recipes)
  53. 1x ligation buffer (see Recipes)
  54. Primer 3 + 4 (10 µM each) (see Recipes)
  55. 6x loading dye (see Recipes)
  56. 50x TAE (see Recipes)

Equipment

  1. Scissors (multiple vendors)
  2. Ice bucket (multiple vendors)
  3. Borosilicate glass bottles (100 ml) (Laborbedarfshop, catalog number: GT00205 )
  4. Borosilicate glass bottles (250 ml) (Laborbedarfshop, catalog number: GT00206 )
  5. Borosilicate glass bottles (500 ml) (Laborbedarfshop, catalog number: GT00207 )
  6. Borosilicate glass bottles (1,000 ml) (Laborbedarfshop, catalog number: GT00208 )
  7. Borosilicate Erlenmeyer flasks (500 ml) (Laborbedarfshop, catalog number: GT00153 )
  8. P1, 10 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641040N )
  9. P10, 100 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641070N )
  10. P20, 200 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641080N )
  11. P100, 1000 µl Finnpipette (Thermo Fisher Scientific, catalog number: 4641100N )
  12. Mortar with pestle with a rough surface for grinding (about 10 cm diameter) (multiple vendors)
  13. Metal spoon (multiple vendors)
  14. Funnels fitting into 50 ml tubes (multiple vendors)
  15. Sieve or tea strainer (about 8 cm diameter) (multiple vendors)
  16. Greenhouse equipped with automatic shading and supplementary light (sodium halogen lamps)
  17. pH meter (multiple vendors)
  18. Water bath (65 °C) (multiple vendors)
  19. -20 °C freezer (multiple vendors)
  20. -80 °C freezer (multiple vendors)
  21. Vortex (multiple vendors)
  22. Measuring cylinder 100 ml, 1 L (multiple vendors)
  23. Autoclave (multiple vendors)
  24. Analytical laboratory balance 'Quintix' (0,1 mg to 120 g) (Sartorius, catalog number: Quintix® 124-1S )
  25. Precision laboratory balance 'Cubis' (10 mg to 2.2 kg) (Sartorius, catalog number: MSE2203P-000-DR )
  26. Ice machine (multiple vendors)
  27. Desiccator (multiple vendors)
  28. Vacuum pump with manometer, condensation trap and tubing to connect desiccator to vacuum pump (multiple vendors)
  29. Fume hood
    Note: All manipulations involving the NIBF buffer must be performed inside a fume hood. Follow the safety regulations of your laboratory during the manipulations and for the waste disposal.
  30. Heraeus Multifuge 4KR centrifuge for 50 ml tubes (3,000 x g required) (Thermo Fisher Scientific, model: HeraeusTM Multifuge 4KR , catalog number: 75004461)
  31. Heraeus Fresco 21 centrifuge for Eppendorf tubes (16,000 x g required) (Thermo Fisher Scientific, model: HeraeusTM FrescoTM 21 , catalog number: 75002426)
  32. Epifluorescence microscope BX61 (Olympus) equipped with a cooled CCD camera (Hamamatsu Orca ER) (see Note 1) (Olympus, model: BX61 )
  33. Incubator cabinet (37 °C) (Memmert, model: Model 600 , catalog number: D06062)
  34. Rocking platform (Heidolph Instruments, model: Titramax 1000, catalog number: 544-12200-00 )
  35. Eppendorf ThermoMixer C (Eppendorf, model: ThermoMixer® C , catalog number: 5382000015) with a Smartblock for 1.5 ml tubes (Eppendorf, catalog number: 5360000038 )
  36. 1 L beaker glass (multiple vendors)
  37. Magnetic stir bar and magnetic stirrer (multiple vendors)
  38. Tube rotator (NeoLab, catalog number: 7-0045 )
  39. Incubator cabinet (16 °C) (GFL-Gesellschaft für Labortechnik, catalog number: 3032 ) placed in a cold room at 4 °C
  40. Qubit 2.0 (or Qubit 4) fluorometer with assay tubes, dsDNA HS Assay and dsDNA BR Assay (Thermo Fisher Scientific, model: Qubit 4, catalog number: Q33227 )
  41. Thermocycler (multiple vendors)
  42. Microwave (multiple vendors)
  43. Agarose gel electrophoresis equipment and accessories [microwave, tray (15 x 15 cm), combs, power supply, electrophoresis buffer, agarose, etc.] (multiple vendors)
  44. Covaris S220 AFA Ultrasonicator (Covaris) and associated equipment [snap-cap microTUBEs (with AFA-fiber and pre-split septum), chiller, software, computer (see Note 1)]
  45. Dark Reader blue light transilluminator (Clare Chemical Research, catalog number: DR46B )
  46. Agilent 2100 Electrophoresis Bioanalyzer or Agilent 4200 TapeStation System (Agilent Technologies, model: Agilent 2100 ) including accessories and consumables (see Note 1)
  47. GenPure Pro UV/UF (Thermo Fisher Scientific, catalog number: 50131950 )

Software

Software and computer hardware:
To analyze Hi-C/TCC sequence data, a computer server running a Unix operating system (e.g., Linux, Solaris, MacOS; see Note 1) or access to a cloud-computing system (e.g., CyVerse, http://www.cyverse.org) is required. Common UNIX command line tools (such as) need to be available. To accelerate CPU-intensive steps such as read alignment, access to a multi-core machine (> 16 CPU cores) is recommended. Depending on the number of samples to be analyzed, hard disk storage space needs to be allocated.
The following bioinformatics software need to be installed to carry out the primary data analysis described below:

  1. Casava, http://support.illumina.com/sequencing/sequencing_software/casava.html
  2. Cutadapt, http://cutadapt.readthedocs.io
  3. BWA-MEM, https://github.com/lh3/bwa
  4. SAMtools, http://www.htslib.org/download/
  5. BEDtools, http://bedtools.readthedocs.io

Procedure

Notes:

  1. All procedures can be performed at room temperature unless specified otherwise.
  2. In order to avoid contaminations of the sequencing libraries, filter tips and gloves should be used.
  3. Surfaces should be cleaned for DNA removal with commercial products at regular intervals.
  4. Lab space and instruments for handling samples pre- and post-PCR must be separated physically.
  5. Although the protocol worked well (> 40 independent experiments), the authors take no liability for the success of the experiments conducted by the reader.

  1. Plant growth and harvesting
    1. About 100 barley seeds are planted in two pots (16 cm diameter) filled with compost soil. Water thoroughly, and grow the plants for 7 days in a greenhouse (sodium halogen lamps, light period of 16 h, night: 18 °C and day: 21 °C). As an example, growth conditions for barley are given in (Zimmermann et al., 2006). Other plant species may require different conditions. Adapt the plant cultivation accordingly. Use young tissues (unexpanded cells) with a favorable content of nuclei.
    2. Grow sufficient plants to obtain about 2.4 g of leaves.
    3. Harvest the leaves with scissors. Wrap the leaves in aluminum foil and store on ice for the transport to the lab until ready for next step.

  2. Chemical crosslinking
    1. Trim the leaves to 0.5-1.0 cm long segments and transfer about 0.8 g tissue into a 50 ml tube. Prepare 3 tubes and store on ice.
    2. Add inside the fume hood 15 ml cold NIBF to each tube. Push a polystyrene plug down to the liquid level for keeping the leaves submersed in the buffer during vacuum infiltration (Hövel et al., 2012) (Figure 2).


      Figure 2. Tube setup during vacuum infiltration. A polystyrene plug fitting snugly to the tube is pushed down to the liquid surface area in order to ensure submersion of the leaf segments during the infiltration.

    3. Place tubes in a desiccator and infiltrate the NIBF (150 mbar). Cut the vacuum abruptly after 5, 10 and 15 min to help the entering of the NIBF into the leaf segments. Continue with the infiltration for a total time of 1 h. The green color of segments will be darker after the infiltration (Figure 3) due to the penetration of the fixative into the intercellular space (Hövel et al., 2012) (see Note 2).


      Figure 3. Leaf color change. The image is taken from a typical leaf segment before (A) and after (B) vacuum infiltration with NIBF buffer (fixative). 

    4. Cut the vacuum, remove the polystyrene plugs and add 2.0 ml of 2 M glycine to stop crosslinking (Hövel et al., 2012). Mix the sample thoroughly by pipetting up and down using a disposable 25 ml plastic pipette. Insert the plugs and apply vacuum (150 mbar) for 5 min.
    5. Dispose the plugs and decant the liquid into a waste container. Use a sieve to keep the leaf segments in the 50 ml tube (Figure 4). Wash the leaves three times by adding 40 ml purified water per tube.
    6. Dry the segments of each tube well with paper towel and store them on ice until grinding (Figure 4).


      Figure 4. Washing and drying of leaf segments after chemical crosslinking. A. Segments are washed with purified water. A sieve is used to withhold the segments in the tube during decantation. B. Leaf segments from each tube (about 0.8 g) are dried thoroughly on a paper towel to facilitate subsequent grinding in liquid nitrogen.

  3. Cell disruption and nuclei isolation
    1. Precool mortar and pestle in liquid nitrogen (see Note 3). Grind about 0.8 g leaves to a fine powder. Use a metal spoon (precooled in liquid nitrogen) to transfer the powder into a pre-cooled 50 ml plastic tube (Figure 5A). Close the tube with a lid, which is punctured several times to ensure pressure balance. Store the material immediately at -80 °C. Prepare the remaining 2 tubes accordingly. Process the samples for nuclei isolation within 1 week.
    2. Place the 3 tubes containing the powder on ice after removing the tubes from the -80° freezer and add 10 ml NIBP (Hövel et al., 2012) to each tube using a 25 ml plastic pipette. Mix carefully with a pipette to resuspend frozen clumps completely.
    3. In order to prevent filters from clogging, distribute the suspension evenly into four funnels prepared in advance (as described below).
    4. Filter the suspension at 4 °C through Miracloth and Sefar Nitex placed in a funnel as described (Hövel et al., 2012). Miracloth should face the plant material and Sefar Nitex the funnel surface. Use gravity flow only to avoid contamination with cell debris. Collect the filtrate in a 50 ml plastic tube (Figure 5B).


      Figure 5. Cell disruption and filtering of nuclei. A. Leaf segments are ground in liquid nitrogen to a fine powder. The material is transferred into a 50 ml tube using a metal spoon and immediately stored at -80 °C. B. For the isolation of nuclei, the powder is resuspended in 10 ml NIBP and filtered through Miracloth and Sefar Nitex. The extract is collected in a clean 50 ml tube for subsequent centrifugation.

    5. Spin the four filtrates in a pre-cooled swing-out centrifuge (3,000 x g, 15 min, 4 °C). Discard the supernatant using a 10 ml plastic pipette and resuspend each pale green pellet in 1 ml ice-cold NIBS (Hövel et al., 2012).
    6. Transfer the suspension to four 1.5 ml tubes and spin in a pre-cooled centrifuge (1,900 x g, 5 min, 4 °C). Discard the supernatant and resuspend the pellets in 300 µl ice-cold NIBS.
    7. Prepare four 2.0 ml tubes each containing 1.5 ml ice-cold sucrose cushion and carefully layer 300 µl of the suspension on top essentially as described (Abdalla et al., 2009) (Figure 6). Spin the four tubes in a pre-cooled centrifuge (16,000 x g, 1 h, 4 °C). Remove the supernatant completely and avoid contaminating the colorless nuclei pellet with the pale green top layer.


      Figure 6. Sucrose cushion. The pale green nuclei extract (e) is layered on top of a sucrose cushion (c) and centrifuged in a 2.0 ml tube for further purification.

    8. Resuspend each of the four nuclei pellets in 100 µl ice-cold NIBS. Pool two nuclei suspensions (200 µl) in a 1.5 ml tube. Place the two pools on ice and perform a microscopic quality check.

  4. Microscopic quality and quantity check of the nuclei (optional)
    Note: The microscopic check might be skipped, if nuclei are purified in a routine manner, or if an epifluorescence microscope is not available.
    1. Stain 7 µl nuclei suspension with 7 µl VECTASHIELD mounting medium with DAPI for the integrity examination (Hövel et al., 2012). Apply the stained sample to a microscopic slide and add the coverslip. Analyze the nuclei using the epifluorescence microscope (100x objective, 1,000-fold magnification, DAPI-filter, absorption 358 nm, emission 461 nm). Intact nuclei appear round or oval and show sharp contours (Figure 7).


      Figure 7. Purified barley nuclei. The integrity of barley nuclei following sucrose cushion centrifugation is examined using epifluorescence microscopy (1,000-fold magnification) and staining with DAPI. The scale bar corresponds to 5 µm.

    2. Stain another aliquot of the nuclei suspension with VECTASHIELD mounting medium with DAPI for quantification. Count the nuclei using a cell counting chamber (Neubauer chamber) and the epifluorescence microscope (20x objective, 200-fold magnification, DAPI-filter, absorption 358 nm, emission 461 nm). Determine the concentration of the nuclei and continue the TCC library construction with approximately 107 nuclei.

  5. Chromatin biotinylation
    1. Spin the two tubes with the nuclei suspension in a pre-cooled centrifuge (1,900 x g, 5 min, 4 °C) and discard the supernatant. Resuspend each pellet in 900 µl ice-cold wash buffer 1 (Kalhor et al., 2011). Spin the sample in a pre-cooled centrifuge (1,900 x g, 5 min, 4 °C) and discard the supernatant.
    2. Repeat the washing step with wash buffer 1.
    3. Resuspend each pellet with wash buffer 1. Each tube should contain a volume of 125 µl sample (total volume of the entire nuclei preparation: 250 µl).
    4. Add 48 µl 2% SDS per tube and incubate in a pre-warmed water bath (65 °C, 10 min) to solubilize the crosslinked chromatin (Kalhor et al., 2011). Cool samples to room temperature.
    5. Add 53 µl 25 mM IPB per tube and agitate in a light-tight container (1 h) to biotinylate the cysteine residues (Kalhor et al., 2011).
    6. For neutralization of SDS add 650 µl 1x NEBuffer 2 per tube and incubate on ice for 5 min (Kalhor et al., 2011).
    7. Add 113 µl 10% Triton X-100 per tube, mix, incubate on ice (10 min) and at 37 °C (10 min) (Kalhor et al., 2011).
    8. The total reaction volume is 1,975 µl (2 tubes with 989 µl each).

  6. HindIII digestion and dialysis
    1. Add 50 µl 10x NEBuffer 2, 2.5 µl 1 M DTT, 215 µl water and 10 µl HindIII (100 U/µl) to each tube (Kalhor et al., 2011).
    2. The total volume per tube is 1,267 µl.
    3. Incubate the two tubes horizontally in an incubator cabinet (37 °C, overnight, gentle agitation on a rocking platform).
    4. Pool the two digestions and inject the sample (about 2.5 ml) into one pre-wetted Slide-A-LyzerTM Dialysis cassette (20 kDa, 3 ml) according to the manufacturer’s instructions. Inject slowly to avoid shearing of the DNA (Figure 8). Aspirate most of the remaining air in the cassette, fill 1 L dialysis buffer into a beaker glass, add a magnetic stir bar and dialyze the cassette inserted into a float buoy with slow agitation for 3 h, thereby removing excess IPB from the biotinylation reaction (Kalhor et al., 2011).


      Figure 8. Dialysis. After the HindIII digestion, the sample (about 2.5 ml) is injected slowly into a pre-wetted Slide-A-LyzerTM Dialysis cassette. Prior to dialysis, most of the air is aspirated from the inside. The cassette is inserted into a float buoy and placed into dialysis buffer for a total of 4 h.

    5. Discard the buffer and dialyze for 1 h in 1 L fresh dialysis buffer.
    6. Collect the DNA as described by the manufacturer of the dialysis cassette. Draw the sample slowly into the syringe to avoid shearing.
    7. Transfer the sample evenly into two 2.0 ml tubes.

  7. Tethering
    1. Prepare MyOne Streptavidin T1 beads (Kalhor et al., 2011). Resuspend the beads by gentle vortexing and dispense 200 µl in each of two 1.5 ml tubes (see Note 4).
    2. Place the tubes into a MPC, wait for 3 min and discard the supernatant.
    3. Remove the tubes from the MPC, add 1 ml PBST per tube and resuspend the beads by gentle vortexing. Place the tubes in the MPC for 3 min and discard the supernatant.
    4. Repeat the washing step with PBST twice.
    5. Resuspend the beads in each tube in 1 ml PBST (total volume 2 ml).
    6. Distribute the dialyzed sample equally into five 1.5 ml tubes and add PBST to a final volume of 500 µl per tube. Label 4 tubes ‘TCC’ and one tube ‘3C’.
    7. Add to each tube 400 µl washed MyOne Streptavidin T1 beads (final volume 900 µl per tube) and place the samples in a tube rotator for 30 min.
    8. During the affinity binding prepare the 25 mM neutralized IPB. Add 7 µl 25 mM neutralized IPB to each of the 5 tubes and place the samples in a tube rotator for 15 min (see Note 5).

  8. Marking DNA ends and circularization
    1. Place the five tubes into an MPC, wait for 2 min and discard the supernatant.
    2. Remove the tubes from the MPC, add 600 µl PBST (Kalhor et al., 2011) per tube and resuspend the beads by gentle vortexing. Place the tubes in the MPC for 2 min and discard the supernatant.
    3. Repeat the washing with 600 µl wash buffer 2 per tube (Kalhor et al., 2011).
    4. Resuspend the beads in 100 µl wash buffer 2 per tube and keep on ice.
    5. Prepare a master mix for marking the DNA ends of the 4 ‘TCC’ samples by adding in the following order (Kalhor et al., 2011):
      295 µl water
      4.5 µl 1 M MgCl2
      45 µl 10x NEBuffer 2
      3.2 µl 10 mM dATP
      3.2 µl 10 mM dTTP
      3.2 µl 10 mM dGTPαS
      68 µl 0.4 mM biotin-14-dCTP
      18 µl 10% Triton X-100
      11.3 µl Klenow DNA polymerase, large fragment (10 U/µl)
      Mix the components gently (see Note 6)
    6. Four ‘TCC’ samples: Add 100 µl of the master mix to the beads resuspended in 100 µl wash buffer 2.
      ‘3C’ control: Add 100 µl 1x NEBuffer 2 buffer (no fill-in reaction and no marking of DNA ends).
    7. If no 3C control for biotin incorporation is desired, use the remainder 3C material for an additional, fifth TCC aliquot. Adjust the volumes of master mixes accordingly.
    8. Place the five samples into a tube rotator for 40 min.
    9. Stop the fill-in reaction by adding 5 µl 0.5 M EDTA, pH 8.0 to each tube (Kalhor et al., 2011) and mix well by inversion.
    10. Place the five tubes into an MPC, wait for 2 min and discard the supernatant.
    11. Remove the tubes from the MPC, add 600 µl wash buffer 3 (Kalhor et al., 2011) per tube and resuspend the beads by gentle vortexing. 
    12. Place the tubes in the MPC for 2 min and discard the supernatant.
    13. Repeat the washing with 600 µl wash buffer 3 per tube.
    14. Resuspend the beads in 500 µl wash buffer 3 per tube and transfer the beads to five 15 ml conical tubes (label four tubes ‘TCC’ and one tube ‘3C’).
    15. Prepare a master mix by adding (Kalhor et al., 2011):
      21.1 ml water
      1,350 µl 10x ligation buffer TCC
      972 µl 10% Triton X-100
      540 µl 1 M Tris-HCl, pH 7.4
      270 µl BSA (10 mg/ml)
    16. Add 4,480 µl of the master mix to the five conical tubes (total volume 4,980 µl/ tube).
    17. Add 12 µl T4 DNA ligase (5 U/µl) to each of the four TCC tubes and 3 µl T4 DNA ligase (5 U/µl) to the ‘3C’ control.
    18. Mix the components gently by inversion.
    19. Incubate horizontally in a cabinet (16 °C, overnight) with agitation fast enough to keep the beads in suspension.
    20. Stop the ligation by adding 200 µl 0.5 M EDTA, pH 8.0 to each tube (Kalhor et al., 2011) and mix gently by inversion.
    21. Reclaim the beads by mounting the five 15 ml tubes horizontally on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96). Fasten the tubes securely to the magnet by using tape or a rubber strap (Figure 9).


      Figure 9. Recovery of tethered circularization products. Tubes containing the ligation products are mounted on a DynaMag-96 Side Skirted Magnetic Particle Concentrator. The beads are reclaimed, and the supernatant is discarded while the tubes are placed in the magnet.

    22. Incubate for 5 min to reclaim the beads.
    23. Discard the supernatant using a 10 ml pipette.

  9. Reversion of crosslink and DNA extraction
    1. Add 400 µl extraction buffer (Kalhor et al., 2011) to each 15 ml tube and resuspend the beads.
    2. Transfer the beads to five 1.5 ml tubes (four labeled ‘TCC’ and one ‘3C’).
    3. Digest remaining RNA by adding 4 µl RNase A (25 µg/µl) to each 1.5 ml tube following incubation at 37 °C for 45 min.
    4. Add 20 µl Proteinase K (20 µg/µl) per tube, mix gently and incubate in a water bath (65 °C, overnight) (Kalhor et al., 2011).
    5. Add additional 5 µl Proteinase K (20 µg/µl) per tube, mix gently and incubate in a water bath (65 °C, 2 h).
    6. Place the five tubes into an MPC, wait for 2 min and transfer the supernatant containing the DNA into five fresh 1.5 ml tubes.
    7. Extract the DNA by adding 400 µl phenol:CHCl3 to each tube (Kalhor et al., 2011). Shake manually for 1 min. Separate the phases by centrifugation (room temperature, 13,000 x g, 10 min). Transfer the upper aqueous phase into a new 1.5 ml tube (see Note 7).
    8. Repeat the extraction with 400 µl phenol:CHCl3.
    9. Repeat the extraction once with 400 µl CHCl3.
    10. Transfer the upper aqueous phase into a new 2.0 ml tube and determine the volumes.
    11. For DNA precipitation add 1/20 volume 4 M NaCl, 1/100 volume glycogen (5 mg/ml) and 2 volumes ice-cold 100% ethanol to each tube (Kalhor et al., 2011) (see Note 8).
    12. Mix the five tubes well by inversion and incubate for 30 min on ice.
    13. Collect the DNA by centrifugation (4 °C, 13,000 x g, 30 min).
    14. Carefully remove the supernatant without disturbing the translucent DNA pellet.
    15. Add 500 µl ice-cold 80% ethanol and spin the DNA (4 °C, 13,000 x g, 5 min).
    16. Discard the supernatant. Perform a pulse-spin and remove liquid remnants.
    17. Dry the pellet (5 min, room temperature).
    18. Dissolve the five DNA pellets in 30 µl EB per tube. Pool the four TCC samples in one 1.5 ml tube. Precipitate the DNA of the TCC and 3C control by adding 1/10 volume 3 M sodium acetate, pH 5.2 and two volumes ice-cold 100% ethanol.
    19. Mix well by inversion and incubate for 30 min on ice.
    20. Collect the DNA by centrifugation (4 °C, 13,000 x g, 30 min).
    21. Carefully remove the supernatant from the two tubes.
    22. Add 500 µl ice-cold 80% ethanol.
    23. Spin the DNA (4 °C, 13,000 x g, 15 min).
    24. Discard the supernatants. Perform a pulse-spin and remove liquid remnants.
    25. Dry the pellets (5 min, room temperature).
    26. Dissolve the TCC sample in 50 µl EB and the 3C control in 10 µl EB.
    27. Determine the DNA concentration using the Qubit 2.0 (or Qubit 4) fluorometer and the dsDNA BR Assay. The concentration should be 100-800 ng/µl.
    28. The samples can be stored at -20 °C.
    29. Measure the biotin incorporation (Belton et al., 2012) (see Note 9).
      Set up two PCR reactions for the 3C control and the TCC sample:
      240 ng DNA
      15 µl 5x Q5 Hot Start buffer
      1.5 µl 10 mM dNTPs
      3.75 µl 10 mM primer 1 + 2 (10 µM each)
      0.75 µl Q5 Hot Start High-Fidelity DNA Polymerase (2U/µl)
      Add water to a final volume of 75 µl
    30. Amplify for 1 cycle (98 °C for 40 sec, 66 °C for 30 sec, 72 °C for 30 sec) followed by 35 cycles (98 °C for 10 sec, 66 °C for 30 sec, 72 °C for 30 sec) and a final amplification (98 °C for 10 sec, 66 °C for 30 sec, 72 °C for 5 min).
    31. Purify the products using the ‘QIAquick PCR Purification Kit’ according to the manufacturer’s instructions. Elute the PCR product in 23 µl EB.
    32. Set up a control and three digestions for the ‘3C control for TCC’ and ‘TCC’ (Table 1). Incubate at 37 °C (incubator cabinet, overnight).

      Table 1. Digestion setup for the verification of marking and ligation of ends


    33. Add 4 µl 6x loading dye and analyze the products on a standard 2% agarose gel (Green and Sambrook, 2012). Typical results are shown in Figure 10.


      Figure 10. Typical control for marking and ligation of ends. Two close genomic barley HindIII fragments are PCR-amplified across their ligation junction. HindIII-digestion of the amplified 3C junctions, in which HindIII sticky-ends are ligated, yields two fragments of a different size. In contrast, TCC junctions are created from the blunt-end ligation of filled-in and marked HindIII ends. As a result, novel NheI sites are formed and the original HindIII sites are lost (Belton et al., 2012). Typically, approximately 50-80% of the PCR-products are cleaved by NheI. The size of the products is indicated (bp). The gel images are taken from Mascher et al. (2017).

  10. Removal of biotin from non-ligated DNA ends and DNA fragmentation
    1. To 5 µg DNA (Step I28) add 9 µl 10x NEBuffer 1 and water to 84 µl.
    2. Add 6 µl Exonuclease III (100 U/µl).
    3. Mix gently and incubate (41 °C, 1 h) (see Note 10).
    4. Stop the reaction by adding 2 µl 0.5 M EDTA, pH 8.0 and 2.5 µl 4 M NaCl (Kalhor et al., 2011).
    5. Incubate (70 °C, 20 min) and adjust the volume with water to 100 µl.
    6. Shear the DNA to 100-500 bp using a Covaris S220 AFA Ultrasonicator (Duty Factor: 10%, PIP: 175 W, Cycles per burst: 200, Set Mode: Frequency sweeping, Time: 60 sec) and Covaris microTUBES.
    7. Control the DNA size by running 100 ng on a standard 2% agarose gel (Green and Sambrook, 2012). Add another sonication cycle, if the DNA is still too large.
    8. Transfer the sample completely into a 1.5 ml tube. Determine the volume exactly.
    9. Add 1.8 volumes AMPure XP beads (room temperature), mix, incubate (5 min).
    10. Reclaim the beads using a MPC (2 min).
    11. Wash beads for 30 sec with 190 µl 70% ethanol while the tube is placed in the MPC. Discard the supernatant.
    12. Repeat the wash step (Step J11) once.
    13. Air dry beads completely. 
    14. Remove the tube from the MPC and add 52 µl EBT for elution. 
    15. Resuspend the beads, incubate (1 min), place the tube in an MPC (1 min).
    16. Transfer 51 µl to a new tube.
    17. The samples can be stored at -20 °C.

  11. End repair and A-tailing
    1. Set a heat block to 20 °C.
    2. For end repair add to the fragmented, cleaned-up DNA in the following order:
      7 µl 10x Tango
      7 µl 2.5 mM dNTP mix
      0.7 µl 100 mM ATP
      1.5 µl T4 DNA polymerase (5 U/µl)
      2.5 µl T4 polynucleotide kinase (10 U/µl)
      0.3 µl Klenow DNA polymerase, large fragment (10 U/µl)
      The total volume is 70 µl
    3. Mix carefully by pipetting up and down.
    4. Incubate (20°C, 30 min) the sample.
    5. Determine the volume exactly.
    6. Add 1.8 volumes AMPure XP beads (room temperature), mix, incubate (5 min).
    7. Reclaim the beads using an MPC (2 min).
    8. Wash beads for 30 sec with 190 µl 70% ethanol while the tube is placed in the MPC. Discard the supernatant.
    9. Repeat wash step (Step K8) once.
    10. Air dry beads completely.
    11. Remove the tube from the MPC and add 39 µl TLE for elution.
    12. Resuspend the beads, incubate (1 min), place the tube in an MPC (1 min).
    13. Transfer 38 µl to a new tube.
    14. Add an ‘A’ by the addition of:
      5 µl 10x NEBuffer 2
      4 µl 2.5 mM dATP
      3 µl Klenow fragment, 3’→5’ exo- (5 U/µl)
      The total volume is 50 µl (Kalhor et al., 2011)
    15. Mix carefully by pipetting up and down and incubate (37 °C, 30 min) the sample.
    16. During the incubation set a heat block to 65 °C.
    17. Inactivate the enzyme by adding 1 µl 0.5 M EDTA, pH 8.0 and incubation at 65 °C for 20 min. Place the samples on ice immediately.
    18. During the inactivation prepare the Dynabeads MyOne Streptavidin C1.

  12. Biotin pull-down
    1. All subsequent steps are performed in 1.5 ml DNA LoBind tubes.
    2. Vortex Dynabeads MyOne Streptavidin C1 and transfer 10 µl to a 1.5 ml tube.
    3. Add 400 µl TWB, mix by pipetting and incubate the beads on a rocking platform for 3 min.
    4. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    5. Remove the tube from the MPC, add 400 µl TWB and mix by pipetting.
    6. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    7. Resuspend beads in 50 µl 2x BB and add 50 µl of DNA for the affinity purification of biotinylated DNA fragments (Kalhor et al., 2011).
    8. Incubate for 30 min with rotation.
    9. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    10. Resuspend beads in 800 µl 1x BB and transfer the suspension into a new tube.
    11. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    12. Repeat washing step with 1x BB.
    13. Remove the tube from the MPC, add 100 µl 1x ligation buffer and mix by pipetting.
    14. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    15. Remove the tube from the MPC and resuspend the beads in 38.8 µl 1x ligation buffer.

  13. Illumina paired-end adapter ligation and PCR
    1. Set up the ligation by adding to 1.5 ml tube:
      6 µl TruSeq DNA Single Indexes (Set A)
      38.8 µl beads with affinity purified DNA
      1.1 µl 10x T4 DNA ligation buffer
      1.1 µl PEG 4000 (50%)
      1.0 µl water
      2.0 µl T4 DNA ligase (5 U/µl).
      Note: For the selection of ‘TruSeq DNA Indexes’, follow the instructions provided in the ‘TruSeq Library Pooling Guide’ (Illumina, San Diego, California, U.S.A.).
      Mix well by pipetting and incubate for 1 h (22°).
    2. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    3. Resuspend beads in 400 µl TWB and rotate 5 min.
    4. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    5. Repeat washing with 400 µl TWB twice.
    6. Resuspend beads in 200 µl 1x BB.
    7. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    8. Resuspend beads in 200 µl 1x NEBuffer 2.
    9. Reclaim the beads using an MPC (1 min) and discard the supernatant.
    10. Repeat washing with 200 µl 1x NEBuffer 2.
    11. Remove the tube from the MPC.
    12. Resuspend the beads in 20 µl 1x NEBuffer 2.
    13. Transfer the sample to a new 1.5 ml tube and store the bead-bound TCC DNA on ice.
    14. Titrate the optimal number of PCR-cycles (Belton et al., 2012).
      Resuspend the beads and add to a PCR tube:
      1.5 µl bead-bound TCC DNA
      1.3 µl 10 µM primer 3 + 4 (see Note 11)
      5.0 µl 5x Q5 Hot Start buffer
      2.0 µl 2.5 mM dNTP mix
      0.3 µl Q5 Hot Start High-Fidelity DNA Polymerase (2 U/µl)
      14.9 µl water (final volume 25 µl).
    15. Titration PCR: After an initial incubation at 98 °C (30 sec), perform the amplification of the DNA (20 cycles: 98 °C for 10 sec, 68 °C for 30 sec, and 72 °C for 30 sec), followed by a final 5 min extension at 72 °C.
    16. Take 4 µl aliquots after 9, 12, 15 and 20 cycles.
    17. Analyse the aliquots using a standard agarose gel (Figure 11). Estimate the amount of products by comparing to a known DNA quantity from the ladder. Choose the number PCR cycles and numbers of reactions (25 µl volume) to produce approximately 200-600 ng DNA for sequencing (see Note 12). In order to avoid PCR artifacts indicated by changes in size distribution of the PCR products, it is recommended to use at most 15 cycles (Belton et al., 2012). The library shown in Figure 11 yielded sufficient amounts of DNA at 12 cycles.


      Figure 11. Titration PCR. After the indicated number of PCR cycles, 2.4 µl of the reaction (initial volume of PCR: 25 µl) is withdrawn, size-separated using a standard 2% agarose gel and stained with ethidium bromide. The size of the GeneRuler 50 bp DNA ladder (L) is indicated (bp). The 500 bp marker band corresponds to 20 ng DNA.

    18. Resuspend the bead-bound TCC DNA and set up the final PCR with the calculated number of reactions. Perform PCR using the optimal number of cycles.

  14. Reaction clean-up and size selection
    1. Pool all PCR-products in one 1.5 ml tube and place the tube in an MPC.
    2. Wait for 1 min and transfer the supernatant containing the library into a new 1.5 ml tube for clean-up and size selection (Belton et al., 2012).
    3. Determine the volume exactly.
    4. Add 1.8 volumes AMPure XP beads (room temperature), mix, incubate (5 min) and reclaim the beads using an MPC (2 min).
    5. Wash beads for 30 sec with 190 µl 70% ethanol while the tube is placed in the MPC.
    6. Discard the supernatant.
    7. Repeat the wash step (Steps N5-N6) once.
    8. Air dry beads completely.
    9. Remove the tube from the MPC and add 27 µl EBT for elution.
    10. Resuspend the beads and incubate (10 min). Tap the tube every 2 min to resuspend the beads.
    11. Place the tube in an MPC (1 min) and transfer 25 µl to a new tube.
    12. Determine the DNA concentration using the Qubit 2.0 (or Qubit 4) fluorometer and the dsDNA BR Assay. The concentration should be 10-30 ng/µl.
    13. Use a gel-purification to make sure that appropriately sized DNA fragments are used for sequencing (Himmelbach et al., 2014) (see Note 13). Weigh 1 g UltraPure Agarose in a 500 ml Erlenmeyer flask. Add 50 ml 1x TAE buffer and melt the agarose using a microwave until dissolved completely. Cool the liquid to 60 °C, add 5 µl SYBR-Gold (see Note 14), mix gently without forming bubbles and cast the gel using standard electrophoresis equipment. Cover the gel with aluminum foil and let the gel solidify (light-sensitive dye).
    14. Transfer 250 ng GeneRuler 50 bp DNA ladder into a tube, add 4 µl 6x loading dye and adjust the volume to 24 µl using EBT. Prepare the TCC sample (25 µl) by adding 5 µl 6x loading dye.
    15. Remove the comb and place the gel in an electrophoresis chamber filled with 1x TAE buffer. Load the TCC library in two adjacent slots. Apply the standard to one lane and perform electrophoresis for 1 h with 5 V/cm (distance between anode and cathode). Protect the gel from light during separation. Visualize the DNA using a ‘Dark reader’ transilluminator (see Note 15) and take a photograph. Use a clean scalpel and excise the region between 300 and 550 bp (Figure 12).


      Figure 12. Gel-purification of a typical TCC library. Following size-separation (standard 2% agarose gel electrophoresis) and staining with SYBR Gold, the library is revealed using a visible blue light emitting ‘Dark reader transilluminator’. The DNA from the framed area between approximately 300 and 550 bp is extracted for sequencing. The size of the GeneRuler 50 bp DNA ladder (L) is indicated (bp).

    16. Place the gel block in a 2.0 ml tube and determine the volume using a balance (e.g., block of 234 mg corresponds to 234 µl).
    17. Purify the DNA using the ‘MinElute Gel Extraction Kit’ essentially as described by the manufacturer. Add 6 volumes QG buffer and dissolve the gel completely under gentle agitation.
    18. Add one gel volume isopropanol and mix by inversion.
    19. Apply 700 μl of the dissolved gel to a MinElute column. Place the column in a collection tube and spin (16,000 x g, 1 min). Discard the flow-through.
    20. Stepwise add the residual liquid to the column (repeat Step N19).
    21. Add 740 μl PE to wash the column, incubate for 3 min, spin the column (16,000 x g, 1 min) and discard the flow-through (see Note 16).
    22. Turn the column (180°) in the rotor and spin (16,000 x g, 1 min).
    23. Discard the collection tube, place the MinElute column into a clean 1.5 ml tube and for elution add 50 μl EB to the resin.
    24. Incubate for 1 min and spin the column (16,000 x g, 1 min).
    25. Store the eluted TCC library at -20 °C.

  15. Quality controls, quantification and sequencing
    1. Measure the DNA concentration using the Qubit 2.0 (or Qubit 4) fluorometer (ds DNA HS Assay). The concentration should be 10-20 ng/µl.
    2. Digest 80 ng TCC library with NheI to estimate the fraction derived from ligation of biotinylated junctions (Belton et al., 2012). Set up the reaction by adding to a 1.5 ml tube:
      80 ng TCC library
      2 µl 10x Tango buffer
      1 µl NheI (10 U/µl)
      Water to a final volume of 20 µl
      Prepare an uncut control (without NheI digestion). Incubate overnight at 37 °C. Analyse the products with a 2% agarose gel using 2 µl GeneRuler 50 bp DNA ladder as a standard. The smaller size distribution of the digested TCC library provides an estimate for the fraction containing true TCC ligation products (Figure 13).


      Figure 13. Quality control of a final TCC library. After digestion with NheI the TCC library (80 ng) is size-separated (standard 2% agarose gel), stained with ethidium bromide and compared to the uncut control (80 ng). The shift to a smaller size distribution is indicative of the presence of NheI sites, which originate from genuine TCC ligation events (Belton et al., 2012; Mascher et al., 2017). In general, we found that the shift is a secure quality indicator. Libraries without a clear shift should not be sequenced, because they will yield only a very small fraction of useful TCC reads. The size of the GeneRuler 50 bp DNA ladder (L) is indicated (bp).

    3. Record the size profile of the library and determine the average size of the TCC library using the Agilent High Sensitivity DNA Kit with the Agilent 2100 Bioanalyzer (Figure 14).


      Figure 14. Size profile of a typical TCC library. The TCC library is size-fractionated using the Agilent 2100 Bioanalyzer. The average library size (Region 1: 434 bp; including 121 bp Illumina P5 and P7 adapter) corresponds to an average insert size of about 313 bp. LM: lower marker peak. UM: upper marker peak. FU: fluorescence unit. bp: base pairs.

    4. Quantify and sequence the library (paired-end, 2x 100 cycles) using an Illumina system as described (Mascher et al., 2013).

Data analysis

Primary sequence data analysis

  1. Run the Illumina CASAVA pipeline (http://support.illumina.com/sequencing/sequencing_software/casava.html) to obtain deconvoluted read files in FASTQ format.
  2. Trim the reads at the junction site with cutadapt (Martin, 2011) using the adapter sequence 'AAGCTAGCTT'.
  3. Align the trimmed read pairs to an appropriate reference genome with BWA mem (Li, 2013). The two reads should be mapped as single ends by specifying the parameters ‘-S’ and ‘-P’. The parameter ‘-M’ should be used to mark shorter split hits as secondary.
  4. Sort BAM files by reference position for duplicates removal. Then sort the BAM files again by read name to have the two ends of a pair at adjacent rows of the BAM file. One of the tools SAMtools (Li et al., 2009), Picard (http://broadinstitute.github.io/picard/) or Novosort (http://www.novocraft.com/products/novosort/) can be used.
  5. Remove reads with non-unique alignments and discard secondary alignments, unmapped reads and duplicated reads with command 'samtools view -q 10 -F1284'.
  6. Assign reads to restriction fragments with BEDTools (Quinlan and Hall, 2010). You will need a BED file (https://genome.ucsc.edu/FAQ/FAQformat.html#format1) with the positions of restriction fragments (i.e., regions between two HindIII sites) obtained from a HindIII in silico digest of your reference genome sequence. Use the command ‘bedtools pairtobed -bedpe -f 1 -type both -abam’. The resulting BEDPE file is a tab-separated file that can be processed with standard UNIX text processing tools such as AWK or Perl. The assignments of the two ends of a pair to restriction fragments are on adjacent lines.
  7. Use UNIX scripts (e.g., in the AWK language) to calculate the insert size based on the distance of alignment start positions to neighboring HindIII sites. Discard fragments with insert sizes above 500 bp, plot the insert size distribution (Figure 15) and compare it to that obtained with the Agilent Bioanalyzer.


    Figure 15. Typical in silico size profile of a TCC library. The sequence data are mapped to a reference genome and assigned to restriction fragments. The insert size of each sequenced fragments is determined from the distance of alignment start positions to the next HindIII restriction site. The mode of the distribution is indicated by a red line.

Notes

  1. Approach the local sales representative for ordering, performance and accessory items.
  2. If darkening of the leaves is not observed, the fixative might not have entered completely, thus indicating an incomplete crosslink. Optimize the infiltration conditions for example by increasing the vacuum or cutting shorter plant segments.
  3. It is mandatory to wear appropriate protective gear. Consult the Safety Officer of your institution for proper handling of liquid nitrogen.
  4. The MyOne Streptavidin T1 beads provide about 250 cm2 surface per ml. By using 400 µl beads, the purified chromatin is tethered at low density on a large surface of about 100 cm2 (Kalhor et al., 2011). Thereby intramolecular ligation of DNA ends is favored.
  5. Free streptavidin residues are saturated with biotin in order to avoid interference with biotin-14-dCTP, which is used for marking the DNA ends during the following step (Kalhor et al., 2011).
  6. During this reaction blunt DNA ends are generated, which are labeled with a biotinylated cytosine residue located 3’ to a phosphorothioate bond introduced by the incorporation of dGTPαS (Kalhor et al., 2011).
  7. Spin the samples in a centrifuge equilibrated to room temperature. Cold temperatures might result in the formation of a turbid water phase. Avoid contaminating the water phase with protein from the interphase during the transfer.
  8. NaCl is used for DNA precipitation, because the solution contains SDS (Green and Sambrook, 2012). Subsequently, the DNA is precipitated in the presence of sodium acetate to remove traces of sodium chloride, which is inhibitory to Exonuclease III (Hoheisel, 1993).
  9. Ligation of filled-in HindIII sites (AAGCTT) generates sites for the restriction enzyme NheI (GCTAGC). To control for effectiveness of fill-in and blunt-end ligation, a PCR fragment (534 bp) from two adjacent barley HindIII restriction fragments is generated. For amplification, primer 1 and primer 2 are used.
  10. Exonuclease III from E. coli catalyzes the stepwise removal of mononucleotides from 3’-hydroxyl termini of duplex DNA (New England BioLabs; Rogers and Weiss, 1980). Thus, Exonuclease III allows for the removal of biotinylated residues from the non-ligated DNA ends. Because phosphorothioate linkages located 5’ to the biotinylated cytosine residue are not cleaved by Exonuclease III (Putney et al., 1981), true ligation junctions are preserved (Kalhor et al., 2011).
  11. Primers 3 and 4 anneal to the Illumina adapter and allow for the amplification of the library.
  12. In routine experiments 11-12 amplification cycles are sufficient. Eight PCR reactions (25 µl volume per reaction) should yield enough DNA for sequencing. Follow the ‘golden rule’ to sustain the complexity of libraries: Keep the number of cycles low and increase the number of PCR reactions to obtain sufficient library for sequencing (Belton et al., 2012).
  13. The BluePippin from Sage Science is an alternative to standard gel-purifications.
  14. Do not use ethidium bromide stained gels. Ultra-violet radiation used for excitation of ethidium bromide will damage the DNA.
  15. DNA is revealed in agarose gels using SYBR-Gold dye and visible blue light emitted from a 'Dark reader' transilluminator as the excitation source.
  16. Place the columns in the rotor consistently, since the columns will be turned 180° for removing traces of liquid.

Recipes

Notes:

  1. All solutions are prepared using purified water (GenPure Pro UV/UF) and stored at room temperature unless specified otherwise.
  2. Good laboratory practice has to be applied throughout the experiment.
  3. It is especially important to consult the Safety Data Sheets and the laboratory/environmental safety rules of your institution for proper handling the reagents and instruments.

  1. 1 M HEPES, pH 8.0 (100 ml)
    1. Weigh 26.0 g HEPES sodium salt (4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt) and add 70 ml water
    2. Stir and adjust the pH to 8.0 with concentrated HCl using a pH meter. Adjust to 100 ml with water
    3. Sterilize using a 0.2 µm filter and a 50 ml syringe (store solution in darkness at room temperature)
  2. 2 M sucrose (40 ml)
    1. Fill 27.4 g sucrose into a 50 ml tube
    2. Add water to a volume of 38 ml and place the 50 ml tube in a warm water bath 
    3. Vortex the warm mixture in regular intervals to dissolve the sucrose completely
    4. Adjust the volume to 40 ml and store the solution at -20 °C
    5. Thaw in a warm water bath prior to use
  3. 1 M KCl (100 ml)
    1. Dissolve 7.5 g KCl in water to a final volume of 100 ml
    2. Use a graduated cylinder to adjust the volume and sterilize by autoclaving
    3. Store at room temperature
  4. 10% (v/v) Triton X-100 (45 ml)
    1. Add 5 ml Triton X-100 to 45 ml water into a 50 ml tube
    2. Mix gently to avoid foaming
    3. Store 10% Triton X-100 and solutions containing Triton X-100 protected from light at room temperature
  5. Nuclei Isolation Buffer (NIBF) with formaldehyde (Hövel et al., 2012) (200 ml)
    Add all of the components into a 250 ml bottle and store on ice before use:
    4 ml 1 M HEPES, pH 8.0
    25 ml 2 M sucrose
    200 µl 1 M MgCl2
    1 ml 1 M KCl
    100.8 g 100% (v/v) glycerol (corresponding to 80 ml)
    5 ml 10% (v/v) Triton X-100
    10.7 ml 37% (w/v) formaldehyde
    200 µl 100 mM PMSF
    0.1% (v/v) 2-mercaptoethanol
    Add water to a final volume of 200 ml
    Note: PMSF inhibits serine proteases and is unstable in water. Due to the short half-life of 35 min at pH 8.0 (James, 1978), PMSF must be added immediately before use. In the air formaldehyde slowly oxidizes to formic acid. Buy small quantities and use them shortly after purchase. Poor quality formaldehyde will spoil the experiment. Store formaldehyde at room temperature, because in the cold a precipitate may be formed. Formaldehyde, PMSF and 2-mercaptoethanol are added inside the fume hood just before use. Stir thoroughly and chill the solution on ice. Use NIBF immediately after composition for vacuum infiltration. 100% glycerol (density 1.26 g/ml) is viscous and sticks to pipettes and measuring cylinders. For convenience, weigh 100.8 g 100% glycerol corresponding to 80 ml in the glass bottle.
  6. 2 M glycine (40 ml)
    1. Weigh 6.0 g glycine into a 50 ml tube and adjust to 40 ml with water
    2. Store at -20 °C and thaw in a warm water bath prior to use
  7. 1 mg/ml Aprotinin (Haring et al., 2007)
    1. Dissolve 1 mg Apronitin in 1 ml water
    2. Store in aliquots at -20 °C 
  8. 1 mg/ml Leupeptin (Haring et al., 2007)
    1. Dissolve 1 mg Leupeptin in 1 ml water
    2. Store in aliquots at -20 °C 
  9. 1 mg/ml Pepstatin (Haring et al., 2007)
    1. Dissolve 1 mg Pepstatin in 1 ml water
    2. Store in aliquots at -20 °C 
  10. Nuclei Isolation Buffer with protease inhibitors (NIBP) (Hövel et al., 2012) (150 ml)
    Add all of the components into a 250 ml bottle and store on ice before use:
    3 ml 1 M HEPES, pH 8.0
    18.8 ml 2 M sucrose
    150 µl 1 M MgCl2
    750 µl 1 M KCl
    75.6 g 100% (v/v) glycerol (corresponding to 60 ml)
    3.8 ml 10% (v/v) Triton X-100
    150 µl 100 mM PMSF
    150 µl 100% (v/v) 2-mercaptoethanol
    150 µl 1 mg/ml Aprotinin
    150 µl 1 mg/ml Leupeptin
    150 µl 1 mg/ml Pepstatin
    Add water to a final volume of 150 ml
    Note: PMSF and 2-mercaptoethanol are added inside the fume hood just before use. Stir thoroughly and chill the solution on ice. Use NIBP immediately after composition. 100% glycerol (density 1.26 g/ml) is viscous and sticks to pipettes and measuring cylinders. For convenience, weigh 75.6 g 100% glycerol corresponding to 60 ml in the glass bottle.
  11. Sucrose cushion (20 ml)
    Add all of the components into a 50 ml plastic tube and store on ice before use:
    400 µl 1 M HEPES, pH 8.0
    16.9 ml 2 M sucrose
    20 µl 1 M MgCl2
    100 µl 1 M KCl
    500 µl 10% (v/v) Triton X-100
    20 µl 100 mM PMSF
    20 µl 100% (v/v) 2-mercaptoethanol
    20 µl 1 mg/ml Aprotinin
    20 µl 1 mg/ml Leupeptin
    20 µl 1 mg/ml Pepstatin
    Add water to a final volume of 20 ml
    Note: PMSF and 2-mercaptoethanol are added inside the fume hood just before use. Stir thoroughly and chill the solution on ice. Use the sucrose cushion immediately after composition.
  12. Nuclei Isolation Buffer with 1.5 M sucrose (NIBS) (20 ml)
    Add all of the components into a 50 ml plastic tube and store on ice before use:
    400 µl 1 M HEPES, pH 8.0
    14.9 ml 2 M sucrose
    20 µl 1 M MgCl2
    100 µl 1 M KCl
    500 µl 10% (v/v) Triton X-100
    20 µl 100 mM PMSF
    20 µl 100% (v/v) 2-mercaptoethanol
    20 µl 1 mg/ml Aprotinin
    20 µl 1 mg/ml Leupeptin
    20 µl 1 mg/ml Pepstatin
    Add water to a final volume of 20 ml
    Note: PMSF and 2-mercaptoethanol are added inside the fume hood just before use. Stir thoroughly and chill the solution on ice. Use NIBS immediately after composition.
  13. 2% (w/v) SDS (100 ml)
    1. Dissolve 2 g SDS (Sodium Dodecyl Sulfate) in 70 ml water
    2. Mix gently, avoid foaming and use a graduated cylinder to adjust the volume to 100 ml
    3. The solution can be stored at room temperature
  14. 4 M NaCl (100 ml)
    1. Dissolve 23.34 g NaCl in 80 ml water
    2. Use a graduated cylinder to adjust the volume to 100 ml, sterilize by autoclaving and store at room temperature
  15. Wash buffer 1 (50 ml)
    Add all of the components into a 50 ml plastic tube:
    2.5 ml 1 M Tris-HCl, pH 8.0
    625 µl 4 M NaCl
    100 µl 0.5 M EDTA, pH 8.0
    Adjust with water to a final volume of 50 ml (Kalhor et al., 2011)
  16. 25 mM IPB (150 µl)
    Dissolve 2 mg EZlink Iodoacetyl-PEG2-Biotin (IPB) in wash buffer 1 (final volume 150 µl)
    Note: Prepare the solution freshly before use. IPB is moisture and light sensitive. Store the reagent at 4-8 °C desiccated in a closed dark container together with dry silicate beads. Prior to opening, equilibrate the vial to room temperature to prevent the condensation of moisture in the vial.
  17. 10x NEBuffer 2 (New England BioLabs) (10 ml)
    Add all of the components:
    1.25 ml 4 M NaCl
    1 ml 1 M Tris-HCl, pH 8.0
    1 ml 1 M MgCl2
    100 µl 1 M DTT
    Add water to 10 ml and store in aliquots at -20 °C
  18. 1x NEBuffer 2
    Prepare by diluting 1 ml 10x NEBuffer 2 stock with 9 ml water
    Store aliquots at -20 °C
  19. HindIII (10 U/µl)
    Add all of the components on ice and mix gently:
    0.5 µl HindIII (100 U/µl)
    4.5 µl 1x NEBuffer 2
    Prepare freshly before use
  20. Dialysis buffer (TE buffer) (2 L)
    Add all of the components:
    20 ml 1 M Tris-HCl, pH 8.0
    4 ml 0.5 M EDTA, pH 8.0
    Add water to final volume of 2 L
  21. Phosphate Buffered Saline with Tween 20, pH 7.4 (PBST) (1 L)
    Add all of the components:
    8.0 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Add water to 800 ml
    Adjust the pH to 7.4 using HCl (Green and Sambrook, 2012)
    Add 1 ml 10% Tween 20 and water to a final volume of 1 L
  22. 25 mM neutralized IPB (75 µl)
    1. Dissolve 1 mg EZlink Iodoacetyl-PEG2-Biotin (IPB) in wash buffer 1 (final volume 75 µl)
    2. Take 40 µl 25 mM IPB and add 7 µl 143 mM 2-mercaptoethanol
    Note: Prepare the solution freshly before use.
  23. 10 mM dATP
    Dilute 100 µl 100 mM dATP with 900 µl water and store at -20 °C
  24. 10 mM dTTP
    Dilute 100 µl 100 mM dTTP with 900 µl water and store at -20 °C
  25. Wash buffer 2 (50 ml) (Kalhor et al., 2011)
    Add all of the components into a 50 ml plastic tube:
    2.5 ml 1 M Tris-HCl, pH 8.0
    625 µl 4 M NaCl
    2 ml 10% (v/v) Triton X-100
    Adjust with water to a final volume of 50 ml 
  26. Wash buffer 3 (50 ml) (Kalhor et al., 2011)
    Add all of the components into a 50 ml plastic tube:
    2.5 ml 1 M Tris-HCl, pH 7.4
    10 µl 0.5 M EDTA, pH 8.0
    2 ml 10% (v/v) Triton X-100
    Adjust with water to a final volume of 50 ml 
  27. 10x ligation buffer TCC (1.5 ml)
    Add all of the components into a 2.0 ml plastic tube:
    750 µl 1 M Tris-HCl, pH 7.5
    150 µl 1 M MgCl2
    150 µl 100 mM ATP
    150 µl 1 M DTT
    300 µl water
    Notes: 
    1. Prepare the solution freshly before use.
    2. The composition is identical to the 10x reaction buffer from New England BioLabs.
  28. 10 mg/ml BSA 
    1. Transfer 200 µl Ultrapure BSA (50 mg/ml) into a tube
    2. Add 800 µl water, mix and store at -20 °C
  29. Extraction buffer (50 ml) (Kalhor et al., 2011)
    Add all of the components:
    2.5 ml 1 M Tris-HCl, pH 8.0
    100 µl 0.5 M EDTA, pH 8.0
    1.25 ml 4 M NaCl
    1 ml 10% (v/v) SDS
    Adjust with water to a final volume of 50 ml 
  30. 25 mg/ml RNase A (1 ml)
    Dissolve 25 mg RNase A (DNase-free) in water in 1 ml final volume and store at 4 °C
  31. 20 mg/ml Proteinase K (150 µl)
    Dissolve 3 mg Proteinase K in 150 µl 10 mM Tris-HCl, pH 8.0
    Prepare solution freshly just before use
  32. 5 mg/ml glycogen (100 µl)
    Dilute 25 µl glycogen (20 mg/ml) with 75 µl water and store at -20 °C
  33. 80% ethanol (100 ml)
    Mix 80 ml 100% ethanol and 20 ml water
    Prepare freshly and store in a tightly closed bottle
    Pre-cool on ice before use.
  34. 70% ethanol (100 ml)
    Mix 70 ml 100% ethanol and 30 ml water
    Prepare freshly and store in a tightly closed bottle
    Pre-cool on ice before use.
  35. 3 M sodium acetate, pH 5.2 (100 ml)
    Prepare 100 ml solution as described in Green and Sambrook (2012).
  36. EB (50 ml)
    Add all of the components:
    500 µl 1 M Tris-HCl, pH 8.0
    49.5 ml water
  37. EBT (50 ml)
    Add all of the components:
    500 µl 1 M Tris-HCl, pH 8.0
    250 µl 10% Tween 20
    49.25 ml water
  38. Primer 1 + 2 (10 µM each) (100 µl)
    Primer 1 (100 µM, 5’- ATCTTCATGCGAGGCAGAGT -3’)
    Primer 2 (100 µM, 5’- ACCGTTGAACCATCTTCAGG -3’)
    Add all of the components:
    10 µl 100 µM primer 1 (100 µM)
    10 µl 100 µM primer 2 (100 µM)
    80 µl water
    Store the primer mix (biotin incorporation control) for barley (Mascher et al., 2017) at -20 °C
  39. 10 mM dNTP mix (1 ml)
    Add 400 µl dNTP mix (25 mM each) to 600 µl water and store at -20 °C
  40. 2.5 mM dNTP mix (1 ml)
    Add 100 µl dNTP mix (25 mM each) to 900 µl water and store at -20 °C
  41. TLE (50 ml)
  42. Tween Wash Buffer (TWB) (50 ml) (Lieberman-Aiden et al., 2009)
    Add all of the components:
    250 µl 1 M Tris-HCl, pH 8.0
    50 µl 0.5 M EDTA, pH 8.0
    250 µl 10% Tween 20
    12.5 ml 4 M NaCl
    Adjust with water to a final volume of 50 ml 
  43. 2x Binding Buffer (2x BB) (50 ml) (Lieberman-Aiden et al., 2009)
    Add all of the components:
    500 µl 1 M Tris-HCl, pH 8.0
    100 µl 0.5 M EDTA, pH 8.0
    25 ml 4 M NaCl
    Adjust with water to a final volume of 50 ml
  44. 1x Binding Buffer (1x BB) (20 ml) (Lieberman-Aiden et al., 2009)
    Dilute 10 ml 2x BB with 10 ml water 
  45. 1x ligation buffer (1 ml)
    Add all of the components:
    90 µl 10x T4 DNA Ligation buffer
    90 µl 50% PEG 4000
    820 µl water
    Store at -20 °C
  46. Primer 3 + 4 (10 µM each) (100 µl)
    Primer 3 (100 µM, 5’- AATGATACGGCGACCACCGAGAT-3’)
    Primer 4 (100 µM, 5’- CAAGCAGAAGACGGCATACGA-3’)
    Add all of the components:
    10 µl 100 µM primer 3 (100 µM)
    10 µl 100 µM primer 4 (100 µM)
    80 µl water
    Store the Illumina library amplification primer mix at -20 °C
  47. 6x loading dye (1 ml)
    Add all of the components:
    700 µl water
    300 µl 100% (v/v) glycerol
    A little bit of Bromophenol Blue
    Note: Add Bromophenol Blue to obtain a light blue color. Overstaining will mask DNA (area between 200-300 bp) in standard agarose gels.
  48. 50x TAE (1 L) (Green and Sambrook, 2012)
    1. Dissolve 242 g Tris base in deionized water
    2. Add 57.1 ml glacial acetic acid and 100 ml 0.5 M EDTA, pH 8.0
    3. Adjust the volume to 1 L and mix thoroughly
    4. The 50x TAE stock is diluted with water to 1x TAE working solution and mixed thoroughly 

Acknowledgments

This work was financially supported by core funding of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, and project funding (‘TRITEX’) from the German Federal Ministry of Education and Research (BMBF, FKZ 0315954) to Nils Stein. The graphic design of Karin Lipfert is gratefully acknowledged. The authors thank Lala Aliyeva-Schnorr for help with microscopic analysis of nuclei. This protocol was adapted for the most part from wet-lab procedures (Hövel et al., 2012; Lieberman-Aiden et al., 2009; Kalhor et al., 2011; Belton et al., 2012) and from bioinformatics procedures described previously (Mascher et al., 2017; Beier et al., 2017). The authors declare no competing interests or conflicts of interest.

References

  1. Abdalla, K. O., Thomson, J. A. and Rafudeen, M. S. (2009). Protocols for nuclei isolation and nuclear protein extraction from the resurrection plant Xerophyta viscosa for proteomic studies. Anal Biochem 384(2): 365-367.
  2. Avni, R., Nave, M., Barad, O., Baruch, K., Twardziok, S. O., Gundlach, H., Hale, I., Mascher, M., Spannagl, M., Wiebe, K., Jordan, K. W., Golan, G., Deek, J., Ben-Zvi, B., Ben-Zvi, G., Himmelbach, A., MacLachlan, R. P., Sharpe, A. G., Fritz, A., Ben-David, R., Budak, H., Fahima, T., Korol, A., Faris, J. D., Hernandez, A., Mikel, M. A., Levy, A. A., Steffenson, B., Maccaferri, M., Tuberosa, R., Cattivelli, L., Faccioli, P., Ceriotti, A., Kashkush, K., Pourkheirandish, M., Komatsuda, T., Eilam, T., Sela, H., Sharon, A., Ohad, N., Chamovitz, D. A., Mayer, K. F. X., Stein, N., Ronen, G., Peleg, Z., Pozniak, C. J., Akhunov, E. D. and Distelfeld, A. (2017). Wild emmer genome architecture and diversity elucidate wheat evolution and domestication. Science 357(6346): 93-97.
  3. Beier, S., Himmelbach, A., Colmsee, C., Zhang, X. Q., Barrero, R. A., Zhang, Q., Li, L., Bayer, M., Bolser, D., Taudien, S., Groth, M., Felder, M., Hastie, A., Simkova, H., Stankova, H., Vrana, J., Chan, S., Munoz-Amatriain, M., Ounit, R., Wanamaker, S., Schmutzer, T., Aliyeva-Schnorr, L., Grasso, S., Tanskanen, J., Sampath, D., Heavens, D., Cao, S., Chapman, B., Dai, F., Han, Y., Li, H., Li, X., Lin, C., McCooke, J. K., Tan, C., Wang, S., Yin, S., Zhou, G., Poland, J. A., Bellgard, M. I., Houben, A., Dolezel, J., Ayling, S., Lonardi, S., Langridge, P., Muehlbauer, G. J., Kersey, P., Clark, M. D., Caccamo, M., Schulman, A. H., Platzer, M., Close, T. J., Hansson, M., Zhang, G., Braumann, I., Li, C., Waugh, R., Scholz, U., Stein, N. and Mascher, M. (2017). Construction of a map-based reference genome sequence for barley, Hordeum vulgare L. Sci Data 4: 170044.
  4. Belton, J. M., McCord, R. P., Gibcus, J. H., Naumova, N., Zhan, Y. and Dekker, J. (2012). Hi-C: a comprehensive technique to capture the conformation of genomes. Methods 58(3): 268-276.
  5. Burton, J. N., Adey, A., Patwardhan, R. P., Qiu, R., Kitzman, J. O. and Shendure, J. (2013). Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol 31(12): 1119-1125.
  6. Dekker, J., Rippe, K., Dekker, M. and Kleckner, N. (2002). Capturing chromosome conformation. Science 295(5558): 1306-1311.
  7. de Wit, E. and de Laat, W. (2012). A decade of 3C technologies: insights into nuclear organization. Genes Dev 26(1): 11-24.
  8. Duan, Z., Andronescu, M., Schutz, K., McIlwain, S., Kim, Y. J., Lee, C., Shendure, J., Fields, S., Blau, C. A. and Noble, W. S. (2010). A three-dimensional model of the yeast genome. Nature 465(7296): 363-367.
  9. Dudchenko, O., Batra, S. S., Omer, A. D., Nyquist, S. K., Hoeger, M., Durand, N. C., Shamim, M. S., Machol, I., Lander, E. S., Aiden, A. P. and Aiden, E. L. (2017). De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science 356(6333): 92-95.
  10. Feng, S., Cokus, S. J., Schubert, V., Zhai, J., Pellegrini, M. and Jacobsen, S. E. (2014). Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis . Mol Cell 55(5): 694-707.
  11. Green, M. and Sambrook, J. (2012). Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, New York.
  12. Grob, S., Schmid, M. W. and Grossniklaus, U. (2014). Hi-C analysis in Arabidopsis identifies the KNOT, a structure with similarities to the flamenco locus of Drosophila. Mol Cell 55(5): 678-693.
  13. Haring, M., Offermann, S., Danker, T., Horst, I., Peterhansel, C. and Stam, M. (2007). Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3: 11.
  14. Himmelbach, A., Knauft, M. and Stein, N. (2014). Plant sequence capture optimised for illumina sequencing. Bio-protocol 4(13): e1166. 
  15. Hoheisel, J. D. (1993). On the activities of Escherichia coli exonuclease III. Anal Biochem 209(2): 238-246.
  16. Hövel, I., Louwers, M. and Stam, M. (2012). 3C Technologies in plants. Methods 58(3): 204-211.
  17. James, G. T. (1978). Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86(2): 574-579.
  18. Kalhor, R., Tjong, H., Jayathilaka, N., Alber, F. and Chen, L. (2011). Genome architectures revealed by tethered chromosome conformation capture and population-based modeling. Nat Biotechnol 30(1): 90-98.
  19. Kaplan, N. and Dekker, J. (2013). High-throughput genome scaffolding from in vivo DNA interaction frequency. Nat Biotechnol 31(12): 1143-1147.
  20. Li, H. (2013). Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiV: 1303.3997.
  21. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R. and Genome Project Data Processing, S. (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25(16): 2078-2079.
  22. Lieberman-Aiden, E., van Berkum, N. L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., Lajoie, B. R., Sabo, P. J., Dorschner, M. O., Sandstrom, R., Bernstein, B., Bender, M. A., Groudine, M., Gnirke, A., Stamatoyannopoulos, J., Mirny, L. A., Lander, E. S. and Dekker, J. (2009). Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326(5950): 289-293.
  23. Liu, C., Wang, C., Wang, G., Becker, C., Zaidem, M. and Weigel, D. (2016). Genome-wide analysis of chromatin packing in Arabidopsis thaliana at single-gene resolution. Genome Res 26(8): 1057-1068.
  24. Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal v. 17, n. 1, p. pp. 10-12, may. ISSN 2226-6089.
  25. Mascher, M., Richmond, T. A., Gerhardt, D. J., Himmelbach, A., Clissold, L., Sampath, D., Ayling, S., Steuernagel, B., Pfeifer, M., D'Ascenzo, M., Akhunov, E. D., Hedley, P. E., Gonzales, A. M., Morrell, P. L., Kilian, B., Blattner, F. R., Scholz, U., Mayer, K. F., Flavell, A. J., Muehlbauer, G. J., Waugh, R., Jeddeloh, J. A. and Stein, N. (2013). Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond. Plant J 76(3): 494-505.
  26. Mascher, M., Gundlach, H., Himmelbach, A., Beier, S., Twardziok, S. O., Wicker, T., Radchuk, V., Dockter, C., Hedley, P. E., Russell, J., Bayer, M., Ramsay, L., Liu, H., Haberer, G., Zhang, X. Q., Zhang, Q., Barrero, R. A., Li, L., Taudien, S., Groth, M., Felder, M., Hastie, A., Simkova, H., Stankova, H., Vrana, J., Chan, S., Munoz-Amatriain, M., Ounit, R., Wanamaker, S., Bolser, D., Colmsee, C., Schmutzer, T., Aliyeva-Schnorr, L., Grasso, S., Tanskanen, J., Chailyan, A., Sampath, D., Heavens, D., Clissold, L., Cao, S., Chapman, B., Dai, F., Han, Y., Li, H., Li, X., Lin, C., McCooke, J. K., Tan, C., Wang, P., Wang, S., Yin, S., Zhou, G., Poland, J. A., Bellgard, M. I., Borisjuk, L., Houben, A., Dolezel, J., Ayling, S., Lonardi, S., Kersey, P., Langridge, P., Muehlbauer, G. J., Clark, M. D., Caccamo, M., Schulman, A. H., Mayer, K. F. X., Platzer, M., Close, T. J., Scholz, U., Hansson, M., Zhang, G., Braumann, I., Spannagl, M., Li, C., Waugh, R. and Stein, N. (2017). A chromosome conformation capture ordered sequence of the barley genome. Nature 544(7651): 427-433.
  27. Metzker, M. L. (2010). Sequencing technologies - the next generation. Nat Rev Genet 11(1): 31-46.
  28. Naumova, N., Imakaev, M., Fudenberg, G., Zhan, Y., Lajoie, B. R., Mirny, L. A. and Dekker, J. (2013). Organization of the mitotic chromosome. Science 342(6161): 948-953.
  29. Putney, S. D., Benkovic, S. J. and Schimmel, P. R. (1981). A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo . Proc Natl Acad Sci U S A 78(12): 7350-7354.
  30. Quinlan, A. R. and Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6): 841-842.
  31. Rao, S. S., Huntley, M. H., Durand, N. C., Stamenova, E. K., Bochkov, I. D., Robinson, J. T., Sanborn, A. L., Machol, I., Omer, A. D., Lander, E. S. and Aiden, E. L. (2014). A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159(7): 1665-1680.
  32. Rogers, G. and Weiss, B. (1980). In: Grossman, L. and Moldave, K. (Eds.). [26] Exonuclease III of Escherichia coli K-12, an AP endonuclease. In: Methods in enzymol. 65: 201-211. New York: Academic Press.
  33. Sexton, T., Yaffe, E., Kenigsberg, E., Bantignies, F., Leblanc, B., Hoichman, M., Parrinello, H., Tanay, A. and Cavalli, G. (2012). Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148(3): 458-472.
  34. Wang, C., Liu, C., Roqueiro, D., Grimm, D., Schwab, R., Becker, C., Lanz, C. and Weigel, D. (2015). Genome-wide analysis of local chromatin packing in Arabidopsis thaliana. Genome Res 25(2): 246-256.
  35. Zhou, X., Lowdon, R. F., Li, D., Lawson, H. A., Madden, P. A., Costello, J. F. and Wang, T. (2013). Exploring long-range genome interactions using the WashU Epigenome Browser. Nat Methods 10(5): 375-376.
  36. Zimmermann, G., Baumlein, H., Mock, H. P., Himmelbach, A. and Schweizer, P. (2006). The multigene family encoding germin-like proteins of barley. Regulation and function in Basal host resistance. Plant Physiol 142(1): 181-192.

简介

染色体构象捕获测序(Hi-C)是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加(3C)(Dekker et al。,2002)。 3C的基本工作流程包括(i)通常用甲醛固定完整的染色质,(ii)用限制酶切割固定的染色质,(iii)在稀释条件下重新连接粘性末端,以促进交联片段之间的连接或随机片段之间的那些和(iv)量化基因组基因座对之间的连接事件的数量(de Wit和de Laat,2012)。在最初的3C方案中,通过半定量PCR扩增对应于少量基因组位点(“一对一”)的选定连接接头来测量连接频率(Dekker et al。,2002 )。然后,染色体构象捕获芯片(4C)和染色体构象捕获碳复制(5C)技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C(Lieberman-Aiden et al。,2009)最终将3C与下一代测序相结合(Metzker,2010)。此处,在再连接之前,用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序,并将得到的读数映射到参考基因组,允许以“多对多”方式确定接触概率,其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理(Lieberman-Aiden et al。,2009)。此后,在酵母等其他真核模型物种中进行了类似的努力(Duan et al。,2010), Drosophila (Sexton et al。,2012)和拟南芥(Grob et al。,2014; Wang et al。,2015; Liu et al。< / em>,2016)。 Hi-C的其他用途包括高分辨率染色质环的研究(Rao et al。,2014; Liu et al。,2016),染色质重组的研究细胞周期(Naumova et al。,2013)和突变体个体中染色质组织的差异(Feng et al。,2014)。这里描述的系留构象捕获协议(TCC)(Kalhor et al。,2011)是原始Hi-C方法的变体(Lieberman-Aiden et al。, 2009)并改编为小麦族。

【背景】任何成功的Hi-C实验都应揭示接触概率的距离依赖性衰减:两个位点在三维空间中并置的频率主要取决于它们在线性基因组中的距离(Lieberman-Aiden 等。< / em>,2009)。这一观察推动了Hi-C在物理作图中的应用:全基因组霰弹枪组件的重叠群之间的Hi-C链接数量可以作为它们之间线性距离的代表。然后可以将该距离信息馈送到图算法中以重建沿染色体的支架的线性顺序(Burton 等人,2013; Kaplan和Dekker,2013)。从我们的TCC方法获得的三维邻近信息用于在高质量大麦参考基因组中对基于BAC的超支架进行排序和定向(Beier et al。,2017; Mascher et al。,2017)。对于Emmer基因组装配,TCC信息以类似的方式用于验证导致染色体标度组装(假分子)的支架(Avni 等人,,2017)。

TCC是一种改良的Hi-C方法,其中关键反应(DNA末端标记和环化)是在固相而不是溶液中进行的(Kalhor et al。,2011)。实际上,在人类细胞中,束缚方法产生了改善的信噪比,从而导致更好的低频相互作用映射(Kalhor et al。,2011)。这一观察结果促使我们为小麦开发了一个类似的方案,该方案基于从幼叶中分离交联核(Hövel et al。,2012),然后是TCC(Kalhor 等。 ,2011)(图1)。简而言之,通过使用甲醛的化学交联(Hövel等人,2012)在天然叶子中捕获染色体构象,其共价连接蛋白质与DNA以及蛋白质彼此。纯化细胞核,染色质中含有的蛋白质的半胱氨酸残基被生物素化(Kalhor et al。,2011)。用限制酶( Hind III)消化DNA,然后通过交联的生物素化蛋白以低密度固定在链霉抗生物素蛋白包被的珠子上(Kalhor et al。,2011) 。填充DNA末端,用生物素标记并环化,同时拴在珠子上(Kalhor 等人,,2011)。交联是相反的,连接连接被亲和纯化并提供用于Illumina测序的衔接子以发现最初捕获的真实的染色体内相互作用位点对(Kalhor 等人,,2011)。

在基因组序列组装的背景下分析染色体构象捕获序列数据。如果可获得染色体尺度参考基因组,则可以将Hi-C(或TCC)读数与其对齐并分配至来自基因组序列的预测的 in silico 的限制性片段。随后,可以量化连接限制对的Hi-C读数的数量,并且计数在较大的基因组窗口(例如,1 Mb箱)中聚集,以获得对之间的接触概率的估计。基因组位点(Lieberman-Aiden et al。,2009)。这些接触概率可用于研究染色质组织及其与各种生物学参数的相互作用,如细胞周期阶段(Naumova et al。,2013)或表观基因组学(Zhou et al 。,2013)。染色体构象捕获测序也可用于从片段化序列组装中重建连续的染色体规模基因组参考序列。 Hi-C / TCC读数被映射到由无序支架组成的序列组装,并且支架之间的链接被计数并用作基因组距离的量度。线性基因组中两个序列支架越接近,连接它们的Hi-C读取对的数量越多。然后该距离信息可用于推导序列支架的线性顺序并使相邻支架相对于彼此定向(Burton 等人,2013; Kaplan和Dekker,2013)。这些方法已应用于昆虫的序列组装(Dudchenko et al。,2017)和小麦基因组(Avni et al。,2017; Mascher et al 。,2017)。


图1.小麦TCC的示意图。收获7天大的植物(I)的叶子用于染色质的化学交联(II)。通过在蛋白质(灰色椭圆形)之间以及蛋白质和DNA(线)之间形成共价键来捕获染色质结构。橙色和浅蓝色区段例示了位于染色体上的两个 Hind III / III片段。细胞核被纯化(III),蛋白质被生物素化(IV,绿色五边形),用于随后将复合物束缚成固相。 DNA被消化(剪刀)

关键字:构象捕获, Hi-C, 物理图谱, 染色质互作, 小麦族, 测序, 基因组组装, 支架

材料和试剂

注意:

  1. 我们在独立实验中测试了所有组件。但是,这个清单并不意味着其他制造商的替代产品也不能表现得那么好。
  2. 使用已发布的设备和化学品并未表明任何竞争利益。

  1. 堆肥土(多个供应商)
  2. 花盆(直径16厘米)(多个供应商)
  3. 铝箔(多个供应商)
  4. 10μl蓝宝石低保留过滤器(Greiner Bio One International,目录号:771265)
  5. 200μl蓝宝石低保留过滤器(Greiner Bio One International,目录号:737265)
  6. 1,000μl蓝宝石低保留过滤器(Greiner Bio One International,目录号:750265)
  7. 0.2μm过滤器,Millex GP(默克,目录号:SLGP033RS)
  8. 10毫升和50毫升注射器(多个供应商)
  9. 50毫升猎鹰管(康宁,目录号:352070)
  10. 适用于干燥器(多个供应商)的50毫升Falcon管架。
  11. 一次性10毫升和25毫升塑料移液器(多个供应商)
  12. 移液器填料(BRAND,目录号:25315)
  13. 纸巾(多个供应商)
  14. 适用于液氮的防护装备(多个供应商)
  15. 用于运输和储存液氮的杜瓦瓶(多个供应商)
  16. Miracloth(默克,目录号:475855-1R)
  17. Sefar Nitex 03-55 / 32(55μm网孔,32%开口面积)(StefanKastenmüllerGmbH)
  18. 管(1.5毫升和2.0毫升; SARSTEDT,目录号:72.690.001和72.695.500)
  19. 显微镜片(76 x 26 x 1 mm)(多个供应商)
  20. 封面(多个供应商)
  21. 细胞计数室(Neubauer室)和玻璃盖(Celeromics)
  22. 针(0.9 x 40 mm,HSW FINE-JECT)(Henke-Sass,Wolf,目录号:8300025263)
  23. Slide-A-Lyzer TM 带浮标的透析盒(0.5-3 ml,截止值20 kDa)(Thermo Fisher Scientific,目录号:66003)
  24. Parafilm(BRAND,目录号:701605)
  25. 用于1.5 ml管(DynaMag-2 Magnet)的磁粉颗粒浓缩器(MPC)(Thermo Fisher Scientific,目录号:12321D)
  26. 15毫升锥形管(康宁,目录号:352196)
  27. 橡胶表带或胶带
  28. DynaMag-96侧裙式磁粉颗粒浓缩器(MPC96)(赛默飞世尔科技,目录号:12027)
  29. PCR管,蓝宝石(Greiner Bio One International,目录号:652250)
  30. Snap-cap microTUBE(带AFA纤维和预裂隔膜)(Covaris,目录号:520045)
  31. 一次性手术刀(多个供应商)
  32. 厚度为0.5至1.0厘米的聚苯乙烯塞(自制)紧密地装入50毫升塑料管中
  33. 种子(由实验者提供)
  34. HEPES钠盐(4-(2-羟乙基)哌嗪-1-乙磺酸钠盐)(Carl Roth,目录号:7020.2)
  35. 浓盐酸(Carl Roth,目录号:4625.1)
  36. 蔗糖(Carl Roth,目录号:9097.1)
  37. MgCl 2 •6H 2 O(Sigma-Aldrich,目录号:M2670)
  38. KCl(霍尼韦尔,Fluka,目录号:60130)
  39. Triton X-100(Carl Roth,产品目录号:3051.3)
  40. 100%甘油(Carl Roth,目录号:3783.1)
  41. 苯基甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:93482)
  42. 2-巯基乙醇(14.3 M)(Carl Roth,目录号:4227.1)
  43. 37%(w / v)甲醛溶液(含10-15%甲醇作为稳定剂)(Sigma-Aldrich,目录号:252549)
  44. 甘氨酸(Carl Roth,目录号:3790.1)
  45. 液氮
  46. 抑肽酶(西格玛奥德里奇,目录号:10820)
  47. Leupeptin(Sigma-Aldrich,目录号:L2884)
  48. Pepstatin(Sigma-Aldrich,目录号:P5318)
  49. 带DAPI的VECTASHIELD封固介质(VECTOR Laboratories,目录号:H-1200)
  50. SDS(十二烷基硫酸钠)(Serva,目录号:20765)
  51. 三(羟甲基)氨基甲烷(Tris碱)(Carl Roth,目录号:5429.3)
  52. NaCl(Carl Roth,目录号:9265.2)
  53. EZlink Iodoacetyl-PEG 2 -Biotin(IPB)(Thermo Fisher Scientific,目录号:21334)
  54. 1,4-二硫苏糖醇(DTT)(Carl Roth,目录号:6908.1)
  55. Hind III(100 U /μl)(New England BioLabs,目录号:R0104T)
  56. Nhe I(10 U /μl)(赛默飞世尔科技,目录号:ER0971)
  57. MyOne Streptavidin T1珠子(赛默飞世尔科技,目录号:65601)
  58. Na 2 HPO 4 (Carl Roth,目录号:P030.2)
  59. KH 2 PO 4 (Carl Roth,目录号:3904.1)
  60. 100 mM dATP(Thermo Fisher Scientific,目录号:R0142)
  61. 100 mM dTTP(Thermo Fisher Scientific,目录号:R0172)
  62. 2'-脱氧鸟苷-5' - (α-硫代) - 三磷酸,钠盐(dGTPαS),1:1 R p 和S p 异构体的混合物(Jena Bioscience ,产品目录号:NU-424S)
  63. 生物素-14-dCTP(赛默飞世尔科技,目录号:19518018)
  64. Klenow DNA聚合酶,大片段(10 U /μl)(Thermo Fisher Scientific,目录号:EP0052)
  65. UltraPure BSA(50 mg / ml)(Thermo Fisher Scientific,目录号:AM2616)
  66. T4 DNA连接酶(5U /μl),含10x T4 DNA连接缓冲液和50%PEG 4000(Thermo Fisher Scientific,目录号:EL0011)
  67. RNase A(无DNA酶)(MACHEREY-NAGEL,目录号:740505)
  68. 蛋白酶K(Thermo Fisher Scientific,目录号:25530015)
  69. 苯酚:CHCl 3 :异戊醇(25:24:1)(Carl Roth,目录号:A156.2)
  70. 8-羟基喹啉(Sigma-Aldrich,目录号:H6878)
  71. 异戊醇(Carl Roth,目录号:8930.1)
  72. CHCl 3 (Carl Roth,目录号:3313.2)
  73. 糖原(赛默飞世尔科技,目录号:R0561)
  74. 100%乙醇(Carl Roth,目录号:9065.4)
  75. Q5热启动高保真DNA聚合酶(2 U /μl)和5x Q5热启动缓冲液(New England BioLabs,目录号:M0493S)
  76. 生物素掺入控制(大麦)引物:
    引物1(5'-ATCTTCATGCGAGGCAGAGT-3')
    引物2(5'-ACCGTTGAACCATCTTCAGG-3')
    注:引物经HPLC纯化,0.2μmol合成规模,溶于H 2 O至100μM;例如,Sigma-Aldrich进行合成。
  77. dNTP Set(100 mM溶液)(Thermo Fisher Scientific,目录号:R0181)
  78. dNTP Mix(每种25 mM)(Thermo Fisher Scientific,目录号:R1121)
  79. MinElute凝胶提取试剂盒(QIAGEN,目录号:28606)
  80. Bromophenol Blue(Carl Roth,目录号:A512.2)
  81. 10x NEBuffer 1缓冲液(New England BioLabs,目录号:B7001S)
  82. UltraPure琼脂糖(Thermo Fisher Scientific,Invitrogen TM ,目录号:16500500)
  83. 来自 E. coli 的外切核酸酶III(100 U /μl)(New England BioLabs,目录号:M0206S)
  84. AMPure XP珠子(Beckman Coulter,目录号:A63881)
  85. 10x Tango(赛默飞世尔科技,目录号:BY5)
  86. 100 mM ATP(赛默飞世尔科技,目录号:R0441)
  87. T4 DNA聚合酶(5 U /μl)(Thermo Fisher Scientific,目录号:EP0062)
  88. T4多核苷酸激酶(10U /μl)(Thermo Fisher Scientific,目录号:EK0031)
  89. Klenow DNA聚合酶,大片段(10U /μl)(Thermo Fisher Scientific,目录号:EP0052)
  90. Klenow片段,3'→5'exo-(5 U /μl)(New England BioLabs,目录号:M0212L)
  91. Dynabeads MyOne Streptavidin C1(赛默飞世尔科技,目录号:65001)
  92. DNA LoBind试管(1.5 ml)(Eppendorf,目录号:0030108051)
  93. TruSeq DNA单指标集A(Illumina,目录号:20015960)
  94. 文库扩增引物:
    引物3(5'-AATGATACGGCGACCACCGAGAT-3')
    引物4(5'-CAAGCAGAAGACGGCATACGA -3')
    注:引物经HPLC纯化,0.2μmol合成规模,溶于H 2 O至100μM;例如,Sigma-Aldrich进行合成。
  95. SYBR-Gold(赛默飞世尔科技,目录号:S11494)
  96. GeneRuler 50 bp DNA ladder(Thermo Fisher Scientific,目录号:SM0373)
  97. 冰醋酸(Carl Roth,目录号:3738.5)
  98. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  99. 100%异丙醇(Carl Roth,目录号:6752.4)
注意:按照Green和Sambrook(2012)中的描述准备以下解决方案(#100-#107)。
  1. 1 M Tris-HCl,pH 8.0(100 ml)
  2. 1 M Tris-HCl,pH 7.4(100 ml)
  3. 1 M Tris-HCl,pH 7.5(100 ml)
  4. 0.5 M EDTA,pH 8.0(100 ml)
  5. 1 M DTT(20 ml)
  6. 苯酚:CHCl 3 :含0.1%8-羟基喹啉的异戊醇(25:24:1)
  7. CHCl 3 :异戊醇(24:1)
  8. 1M MgCl 2
  9. 1 M HEPES,pH 8.0(参见食谱)&nbsp;
  10. 2 M蔗糖(见食谱)
  11. 1 M KCl(见食谱)
  12. 10%(v / v)Triton X-100(见食谱)
  13. 含甲醛的细胞核分离缓冲液(NIBF)(见食谱)
  14. 2 M甘氨酸(见食谱)
  15. 1 mg / ml抑肽酶(见食谱)
  16. 1 mg / ml Leupeptin(见食谱)
  17. 1 mg / ml Pepstatin(见食谱)
  18. 具有蛋白酶抑制剂(NIBP)的细胞核分离缓冲液(参见食谱)
  19. 蔗糖垫(见食谱)
  20. 含1.5 M蔗糖(NIBS)的细胞核分离缓冲液(参见食谱)
  21. 2%(w / v)SDS(见食谱)
  22. 4 M NaCl(参见食谱)
  23. 洗涤缓冲液1(见食谱)
  24. 25 mM IPB(见食谱)
  25. 10x NEBuffer 2(见食谱)
  26. 1x NEBuffer 2(见食谱)
  27. Hind III(10 U /μl)(见食谱)
  28. 透析缓冲液(TE缓冲液)(见食谱)
  29. 磷酸盐缓冲盐水,吐温20,pH 7.4(PBST)(见食谱)
  30. 25 mM中和IPB(参见食谱)
  31. 10 mM dATP(见食谱)
  32. 10 mM dTTP(见食谱)
  33. 洗涤缓冲液2(见食谱)
  34. 洗涤缓冲液3(见食谱)
  35. 10x连接缓冲液TCC(见食谱)
  36. 10毫克/毫升BSA(见食谱)
  37. 提取缓冲液(见食谱)
  38. 25 mg / ml RNase A(见食谱)
  39. 20 mg / ml蛋白酶K(见食谱)
  40. 5毫克/毫升糖原(见食谱)
  41. 80%乙醇(见食谱)
  42. 70%乙醇(见食谱)
  43. 3 M醋酸钠,pH 5.2(参见食谱)
  44. EB(见食谱)
  45. EBT(见食谱)
  46. 引物1 + 2(每个10μM)(见食谱)
  47. 10 mM dNTP混合物(参见食谱)
  48. 2.5 mM dNTP混合物(参见食谱)
  49. TLE(见食谱)
  50. 吐温洗涤缓冲液(TWB)(见食谱)
  51. 2x结合缓冲液(2x BB)(见食谱)
  52. 1x结合缓冲液(1x BB)(见食谱)
  53. 1x连接缓冲液(见食谱)
  54. 引物3 + 4(每种10μM)(见食谱)
  55. 6x上样染料(见食谱)
  56. 50x TAE(见食谱)

设备

  1. 剪刀(多个供应商)
  2. 冰桶(多个供应商)
  3. 硼硅酸盐玻璃瓶(100毫升)(Laborbedarfshop,目录号:GT00205)
  4. 硼硅酸盐玻璃瓶(250 ml)(Laborbedarfshop,目录号:GT00206)
  5. 硼硅酸盐玻璃瓶(500毫升)(Laborbedarfshop,目录号:GT00207)
  6. 硼硅酸盐玻璃瓶(1,000毫升)(Laborbedarfshop,目录号:GT00208)
  7. Borosilicate Erlenmeyer烧瓶(500 ml)(Laborbedarfshop,目录号:GT00153)
  8. P1,10μlFinnipepipette(Thermo Fisher Scientific,目录号:4641040N)
  9. P10,100μlFinnpipette(Thermo Fisher Scientific,目录号:4641070N)
  10. P20,200μlFinnpipette(Thermo Fisher Scientific,目录号:4641080N)
  11. P100,1000μlFinnpipette(Thermo Fisher Scientific,目录号:4641100N)
  12. 砂浆用研杵粗糙表面研磨(直径约10厘米)(多个供应商)
  13. 金属勺子(多个供应商)
  14. 漏斗适合50毫升管(多个供应商)
  15. 筛子或滤茶器(直径约8厘米)(多个供应商)
  16. 温室配有自动遮阳和补充灯(钠卤灯)
  17. pH计(多个供应商)
  18. 水浴(65°C)(多个供应商)
  19. -20°C冰柜(多个供应商)
  20. -80°C冰柜(多个供应商)
  21. Vortex(多个供应商)
  22. 量筒100毫升,1升(多个供应商)
  23. 高压灭菌器(多个供应商)
  24. 分析实验室天平'Quintix'(0.1 mg至120 g)(Sartorius,目录号:Quintix ® 124-1S)
  25. 精密实验室天平'Cubis'(10 mg至2.2 kg)(Sartorius,目录号:MSE2203P-000-DR)
  26. 制冰机(多个供应商)
  27. 干燥器(多个供应商)
  28. 带压力计,冷凝水收集器和管道的真空泵将干燥器连接到真空泵(多个供应商)
  29. 通风橱
    注意:所有涉及NIBF缓冲区的操作必须在通风橱内进行。在操作和废物处理过程中,请遵守实验室的安全规定。
  30. 用于50 ml管的Heraeus Multifuge 4KR离心机(需要3,000 x g )(Thermo Fisher Scientific,型号:Heraeus TM Multifuge 4KR,目录号:75004461)
  31. 用于Eppendorf管的Heraeus Fresco 21离心机(需要16,000 xg )(Thermo Fisher Scientific,型号:Heraeus TM Fresco TM 21,目录号:75002426 )
  32. 落射荧光显微镜BX61(奥林巴斯)配备冷却CCD相机(Hamamatsu Orca ER)(参见注1 )(奥林巴斯,型号:BX61)
  33. 培养箱(37°C)(Memmert,型号:600型,目录号:D06062)
  34. 摇摆平台(Heidolph Instruments,型号:Titramax 1000,目录号:544-12200-00)
  35. Eppendorf ThermoMixer C(Eppendorf,型号:ThermoMixer ® C,目录号:5382000015),带有1.5 ml管的Smartblock(Eppendorf,目录号:5360000038)
  36. 1 L烧杯玻璃(多个供应商)
  37. 磁力搅拌棒和磁力搅拌器(多个供应商)
  38. 管旋转器(NeoLab,目录号:7-0045)
  39. 将培养箱(16°C)(GFL-GesellschaftfürLabortechnik,目录号:3032)置于4°C的冷藏室中
  40. 带有分析管的Qubit 2.0(或Qubit 4)荧光计,dsDNA HS Assay和dsDNA BR Assay(Thermo Fisher Scientific,型号:Qubit 4,目录号:Q33227)
  41. 热循环仪(多个供应商)
  42. 微波炉(多个供应商)
  43. 琼脂糖凝胶电泳设备及配件[微波炉,托盘(15 x 15 cm),梳子,电源,电泳缓冲液,琼脂糖,等等)<(多个供应商)
  44. Covaris S220 AFA超声波发生器(Covaris)及相关设备[snap-cap microTUBE(带AFA光纤和预裂隔膜),冷水机,软件,电脑(见注1 )]
  45. Dark Reader蓝光透照仪(Clare Chemical Research,目录号:DR46B)
  46. Agilent 2100电泳生物分析仪或Agilent 4200 TapeStation系统(Agilent Technologies,型号:Agilent 2100),包括附件和消耗品(参见注1 )
  47. GenPure Pro UV / UF(赛默飞世尔科技,目录号:50131950)

软件

软件和计算机硬件:
分析Hi-C / TCC序列数据,运行Unix操作系统的计算机服务器(例如,Linux,Solaris,MacOS;参见注1 )或访问云计算系统(例如,CyVerse, http://www.cyverse.org ) 是必须的。常见的UNIX命令行工具(例如)需要可用。为了加速CPU密集型步骤(例如读取对齐),建议访问多核机器(> 16个CPU内核)。根据要分析的样本数量,需要分配硬盘存储空间。
需要安装以下生物信息学软件来执行下面描述的主要数据分析:

  1. Casava, http://support.illumina.com/sequencing/sequencing_software/casava.html
  2. Cutadapt, http://cutadapt.readthedocs.io
  3. BWA-MEM, https://github.com/lh3/bwa
  4. SAMtools, http://www.htslib.org/download/
  5. BEDtools, http://bedtools.readthedocs.io

程序

注意:

  1. 除非另有说明,否则所有程序均可在室温下进行。
  2. 为避免测序库的污染,应使用过滤嘴和手套。
  3. 应定期清洁表面,以便用商业产品去除DNA。
  4. 用于处理PCR前后样品的实验室空间和仪器必须在物理上分开。
  5. 尽管该协议运作良好(> 40次独立实验),但作者对读者进行的实验成功不承担任何责任。

  1. 植物生长和收获
    1. 将大约100粒大麦种子种植在填充有堆肥土壤的两个盆(直径16cm)中。彻底浇水,在温室中植物生长7天(卤素灯钠灯,光照时间16小时,夜晚:18°C,白天:21°C)。例如,大麦的生长条件在(Zimmermann et al。,2006)中给出。其他植物物种可能需要不同的条件。相应地适应植物栽培。使用具有有利核含量的年轻组织(未扩增细胞)。
    2. 种植足够的植物以获得约2.4g的叶子。
    3. 用剪刀收割树叶。将叶子包裹在铝箔中并储存在冰上以便运输到实验室,直到准备好进行下一步。

  2. 化学交联
    1. 将叶子修剪成0.5-1.0cm长的片段,并将约0.8g组织转移到50ml管中。准备3管并存放在冰上。
    2. 在通风橱内加入15毫升冷的NIBF到每个管中。将聚苯乙烯塞向下推至液位,以便在真空渗透过程中将叶子浸没在缓冲液中(Hövel et al。,2012)(图2)。


      图2.真空渗透过程中的管道安装。将一个紧贴管子的聚苯乙烯塞子向下推到液体表面区域,以确保在渗透过程中浸没叶片段。

    3. 将管放入干燥器中并渗入NIBF(150毫巴)。在5,10和15分钟后突然切断真空,以帮助NIBF进入叶片段。继续浸润总共1小时。由于固定剂渗入细胞间隙,渗透后(图3)的片段绿色会变暗(Hövel et al。,2012)(见注2))。


      图3.叶片颜色变化图像取自典型叶片段(A)之前和(B)真空渗入NIBF缓冲液(固定剂)。&nbsp;

    4. 切开真空,取出聚苯乙烯塞,加入2.0毫升2M甘氨酸以停止交联(Hövel et al。,2012)。使用一次性25毫升塑料移液管上下移液,彻底混合样品。插入塞子并施加真空(150毫巴)5分钟。
    5. 处理塞子并将液体倒入废物容器中。用筛子将叶片保持在50毫升管中(图4)。每管加入40ml纯净水洗净叶子三次。
    6. 用纸巾擦干每个管段,并将其存放在冰上直至研磨(图4)。


      图4.化学交联后洗涤和干燥叶片段。 A.用纯净水洗涤切片。在倾析期间,使用筛子来保留管中的区段。 B.将来自每个管(约0.8g)的叶片在纸巾上彻底干燥,以便随后在液氮中研磨。

  3. 细胞破裂和细胞核分离
    1. 在液氮中预冷研钵和研杵(见注3 )。研磨约0.8克叶子至细粉末。使用金属勺(在液氮中预冷)将粉末转移到预先冷却的50毫升塑料管中(图5A)。用盖子盖住管子,将其刺破几次以确保压力平衡。立即将材料储存在-80°C。相应地准备剩余的2个管。在1周内处理样品以进行细胞核分离。
    2. 从-80°冰箱中取出试管后,将含有粉末的3管放在冰上,用25ml塑料移液管将10ml NIBP(Hövel等,2012)加入各管中。用移液管小心混合,完全重悬冷冻块。
    3. 为了防止过滤器堵塞,将悬浮液均匀地分配到预先准备的四个漏斗中(如下所述)。
    4. 如上所述(Hövel et al。,2012),将悬浮液在4℃下通过Miracloth和Sefar Nitex过滤器置于漏斗中。 Miracloth应该面对植物材料而Sefar Nitex应该面对漏斗表面。仅使用重力流动以避免细胞碎片污染。将滤液收集在50毫升塑料管中(图5B)。


      图5.细胞破碎和细胞核过滤。 A.将叶片在液氮中研磨成细粉。使用金属勺将材料转移到50ml管中并立即储存在-80℃。 B.为了分离细胞核,将粉末重悬于10ml NIBP中,并通过Miracloth和Sefar Nitex过滤。将提取物收集在干净的50ml管中,用于随后的离心。

    5. 在预冷却的摆出式离心机(3,000 x g ,15分钟,4℃)中旋转四种滤液。使用10ml塑料移液管丢弃上清液,并将每个淡绿色沉淀重悬于1ml冰冷的NIBS中(Hövel et al。,2012)。
    6. 将悬浮液转移到四个1.5ml管中并在预冷却的离心机中旋转(1,900 x g ,5分钟,4℃)。弃去上清液,将沉淀重悬于300μl冰冷的NIBS中。
    7. 制备四个2.0ml管,每个管含有1.5ml冰冷的蔗糖垫,并且基本上如所述(Abdalla 等人,2009)(图6),在顶部小心地铺上300μl悬浮液。在预冷却的离心机中旋转四个管(16,000 x g ,1小时,4℃)。完全去除上清液,避免用淡绿色顶层污染无色核颗粒。


      图6.蔗糖垫。 将浅绿色核提取物(e)层叠在蔗糖垫(c)上,并在2.0 ml管中离心,以进一步纯化。

    8. 将四个核颗粒中的每一个重悬于100μl冰冷的NIBS中。将两个细胞核悬浮液(200μl)置于1.5ml管中。将两个池放在冰上并进行微观质量检查。

  4. 核的微观质量和数量检查(可选)
    注意:如果细胞核以常规方式纯化,或者如果没有落射荧光显微镜,可能会跳过显微镜检查。
    1. 用含有DAPI的7μlVECTASHIELD封固剂染色7μl细胞核悬浮液进行完整性检查(Hövel et al。,2012)。将染色的样品应用于显微镜载玻片并添加盖玻片。使用落射荧光显微镜(100x物镜,1,000倍放大率,DAPI滤光片,吸收358nm,发射461nm)分析核。完整的细胞核呈圆形或椭圆形,并呈现清晰的轮廓(图7)。


      图7.纯化的大麦核。使用落射荧光显微镜(1,000倍放大)检查蔗糖垫离心后的大麦核的完整性,并用DAPI染色。比例尺对应5μm。

    2. 用含有DAPI的VECTASHIELD封固剂染色另一等份的细胞核悬浮液用于定量。使用细胞计数室(Neubauer室)和落射荧光显微镜(20x物镜,200倍放大率,DAPI-滤光片,吸收358nm,发射461nm)计数细胞核。确定细胞核的浓度并继续使用大约10个 7 细胞核进行TCC文库构建。

  5. 染色质生物素化
    1. 在预冷却的离心机(1,900 x g ,5分钟,4℃)中用核悬浮液旋转两个管并弃去上清液。将每个沉淀重悬于900μl冰冷的洗涤缓冲液1中(Kalhor 等人,,2011)。在预冷却的离心机(1,900 x g ,5分钟,4℃)中旋转样品并弃去上清液。
    2. 用洗涤缓冲液1重复洗涤步骤。
    3. 用洗涤缓冲液1重悬每个沉淀。每个试管应含有125μl体积的样品(整个细胞核制剂的总体积:250μl)。
    4. 每管加入48μl2%SDS,并在预热的水浴(65℃,10分钟)中孵育以溶解交联的染色质(Kalhor 等人,,2011)。将样品冷却至室温。
    5. 每管加入53μl25mMIPB,并在不透光的容器中搅拌(1小时)以使半胱氨酸残基生物素化(Kalhor 等人,,2011)。
    6. 对于SDS的中和,每管加入650μl1xNEBuffer 2并在冰上孵育5分钟(Kalhor 等人,,2011)。
    7. 每管加入113μl10%Triton X-100,混合,在冰上孵育(10分钟)和37℃(10分钟)(Kalhor 等人,,2011)。
    8. 总反应体积为1,975μl(2管,每管989μl)。

  6. Hind III消化和透析
    1. 向每个管中加入50μl10xNEBuffer2,2.5μl1M DTT,215μl水和10μl Hind III /(III U /μl)(Kalhor 等。, 2011)。
    2. 每管的总体积为1,267μl。
    3. 将两个试管水平孵育在培养箱中(37°C,过夜,在摇动平台上轻轻搅拌)。
    4. 将两个消化物汇集并根据制造商的说明将样品(约2.5ml)注入一个预先润湿的Slide-A-Lyzer TM 透析盒(20kDa,3ml)中。缓慢注射以避免DNA的剪切(图8)。吸入盒中剩余的大部分空气,将1L透析缓冲液装入烧杯中,加入磁力搅拌棒,将插入浮标的盒子透析,缓慢搅拌3小时,从而从生物素化反应中除去过量的IPB( Kalhor et al。,2011)。


      图8.透析。 Hind III消化后,将样品(约2.5 ml)缓慢注入预先润湿的Slide-A-Lyzer TM < / sup>透析盒。在透析之前,大部分空气从内部被吸出。将盒子插入浮标浮标并放入透析缓冲液中共4小时。

    5. 弃去缓冲液并在1L新鲜透析缓冲液中透析1小时。
    6. 如透析盒的制造商所述收集DNA。将样品缓慢地吸入注射器以避免剪切。
    7. 将样品均匀地转移到两个2.0毫升的管中。

  7. Tethering
    1. 制备MyOne Streptavidin T1珠子(Kalhor et al。,2011)。通过温和涡旋重悬珠子,并在两个1.5ml管中分配200μl(参见注释4 )。
    2. 将试管放入MPC中,等待3分钟并弃去上清液。
    3. 从MPC中取出试管,每管加入1ml PBST,轻轻涡旋重悬珠子。将管置于MPC中3分钟并弃去上清液。
    4. 用PBST重复洗涤步骤两次。
    5. 将每个管中的珠子重悬于1ml PBST(总体积2ml)中。
    6. 将透析的样品均匀地分配到五个1.5ml管中,并将PBST加入到每管500μl的最终体积中。标记4管'TCC'和一管'3C'。
    7. 向每个管中加入400μl洗过的MyOne链霉抗生物素蛋白T1珠(每管最终体积900μl)并将样品置于管旋转器中30分钟。
    8. 在亲和力结合期间,制备25mM中和的IPB。向5个试管中的每一个中加入7μl25mM中和的IPB,并将样品置于管旋转器中15分钟(参见注释5 )。

  8. 标记DNA结束和环化
    1. 将五个试管放入MPC中,等待2分钟并弃去上清液。
    2. 从MPC中取出试管,每管加入600μlPBST(Kalhor 等,2011)并通过温和涡旋重悬珠子。将管置于MPC中2分钟并弃去上清液。
    3. 每管用600μl洗涤缓冲液2重复洗涤(Kalhor 等人,,2011)。
    4. 将珠子重悬于每管100μl洗涤缓冲液2中并保持在冰上。
    5. 通过按以下顺序添加来制备用于标记4'TCC'样品的DNA末端的主混合物(Kalhor et al。,2011):
      295μl水
      4.5μl1MMgCl 2
      45μl10xNEBuffer 2
      3.2μl10mM dATP
      3.2μl10mM dTTP
      3.2μl10mMdGTPαS
      68μl0.4mM生物素-14-dCTP
      18μl10%Triton X-100
      11.3μlKlenowDNA聚合酶,大片段(10 U /μl)
      轻轻混合组分(参见注释6 )
    6. 四个'TCC'样品:将100μl主混合物加入到重悬于100μl洗涤缓冲液2中的珠子中。
      '3C'对照:加入100μl1xNEBuffer 2缓冲液(无填充反应且DNA末端没有标记)。
    7. 如果不需要用于生物素掺入的3C对照,则使用剩余的3C材料用于另外的第五TCC等分试样。相应地调整主混合物的体积。
    8. 将五个样品放入管旋转器中40分钟。
    9. 通过向每个管中加入5μl0.5MEDTA,pH8.0来阻止填充反应(Kalhor 等,,2011)并通过倒置充分混合。
    10. 将五个试管放入MPC中,等待2分钟并弃去上清液。
    11. 从MPC中取出试管,每管加入600μl洗涤缓冲液3(Kalhor 等,2011),轻轻涡旋重悬珠子。&nbsp;
    12. 将管置于MPC中2分钟并弃去上清液。
    13. 每管用600μl洗涤缓冲液3重复洗涤。
    14. 将珠子重新悬浮在每管500μl洗涤缓冲液3中,并将珠子转移到五个15ml锥形管中(标记四个管'TCC'和一个管'3C')。
    15. 通过添加(Kalhor et al。,2011)来准备主混合物:
      21.1毫升水
      1,350μl10x连接缓冲液TCC
      972μl10%Triton X-100
      540μl1MTris-HCl,pH 7.4
      270μlBSA(10 mg / ml)
    16. 将4,480μl主混合物添加到五个锥形管中(总体积4,980μl/管)。
    17. 将12μlT4DNA连接酶(5U /μl)加入到四个TCC管中的每一个中,并将3μlT4DNA连接酶(5U /μl)加入'3C'对照中。
    18. 通过倒置轻轻混合组分。
    19. 在柜中水平孵育(16°C,过夜),搅拌速度足以使珠子保持悬浮状态。
    20. 通过向每个管中加入200μl0.5MEDTA,pH8.0来阻止连接(Kalhor 等,,2011)并通过倒置轻轻混合。
    21. 通过将五个15ml管水平安装在DynaMag-96 Side Skirted Magnetic Particle Concentrator(MPC96)上来回收珠子。使用胶带或橡皮带将管牢固地固定在磁铁上(图9)。


      图9.系留环化产物的回收。将含有连接产物的试管安装在DynaMag-96 Side Skirted Magnetic Particle Concentrator上。回收珠子,弃去上清液,同时将管子放入磁铁中。

    22. 孵育5分钟以回收珠子。
    23. 使用10ml移液管丢弃上清液。

  9. 交联和DNA提取的逆转
    1. 向每个15ml管中加入400μl提取缓冲液(Kalhor et al。,2011)并重悬珠。
    2. 将珠子转移到五个1.5ml管(四个标记为'TCC'和一个'3C')。
    3. 在37℃温育45分钟后,通过向每个1.5ml管中加入4μlRNaseA(25μg/μl)消化剩余的RNA。
    4. 每管加入20μl蛋白酶K(20μg/μl),轻轻混匀,在水浴中孵育(65°C,过夜)(Kalhor et al。,2011)。
    5. 每管加入额外的5μl蛋白酶K(20μg/μl),轻轻混合并在水浴(65°C,2小时)中孵育。
    6. 将5个试管放入MPC中,等待2分钟,将含有DNA的上清液转移到5个新鲜的1.5ml试管中。
    7. 通过向每个管中加入400μl苯酚:CHCl 3 来提取DNA(Kalhor 等人,,2011)。手动摇动1分钟。通过离心分离各相(室温,13,000 x g ,10分钟)。将上层水相转移到新的1.5 ml管中(参见注7 )。
    8. 用400μl苯酚:CHCl 3 重复萃取。
    9. 用400μlCHCl 3 重复萃取一次。
    10. 将上层水相转移到新的2.0ml管中并测定体积。
    11. 对于DNA沉淀,向每个管中加入1/20体积的4M NaCl,1/100体积的糖原(5mg / ml)和2体积的冰冷的100%乙醇(Kalhor 等。,2011) (见注8 )。
    12. 通过倒置将五个管充分混合并在冰上孵育30分钟。
    13. 通过离心(4℃,13,000 x g ,30分钟)收集DNA。
    14. 小心去除上清液,不要打扰半透明的DNA沉淀。
    15. 加入500μl冰冷的80%乙醇并旋转DNA(4℃,13,000 x g ,5分钟)。
    16. 丢弃上清液。进行脉冲旋转并去除液体残留物。
    17. 将颗粒干燥(5分钟,室温)。
    18. 将每个管中的5个DNA颗粒溶解在30μlEB中。将四个TCC样品放入一个1.5 ml管中。通过加入1/10体积的3M乙酸钠,pH 5.2和两倍体积的冰冷的100%乙醇沉淀TCC和3C对照的DNA。
    19. 通过倒置充分混合并在冰上孵育30分钟。
    20. 通过离心(4℃,13,000 x g ,30分钟)收集DNA。
    21. 小心地从两个管中取出上清液。
    22. 加入500μl冰冷的80%乙醇。
    23. 旋转DNA(4℃,13,000 x g ,15分钟)。
    24. 丢弃上清液。进行脉冲旋转并去除液体残留物。
    25. 干燥颗粒(5分钟,室温)。
    26. 将TCC样品溶解于50μlEB中,将3C对照溶解于10μlEB中。
    27. 使用Qubit 2.0(或Qubit 4)荧光计和dsDNA BR Assay确定DNA浓度。浓度应为100-800 ng /μl。
    28. 样品可以在-20°C下储存。
    29. 测量生物素掺入量(Belton et al。,2012)(参见注释9 )。
      为3C对照和TCC样品设置两个PCR反应:
      240 ng DNA
      15μl5xQ5热启动缓冲区
      1.5μl10mM dNTPs
      3.75μl10mM引物1 + 2(各10μM)
      0.75μlQ5热启动高保真DNA聚合酶(2U /μl)
      加水至最终体积为75μl
    30. 放大1个循环(98°C,40秒,66°C,30秒,72°C,30秒),然后进行35个循环(98°C,10秒,66°C,30秒,72°C,30秒) sec)和最终扩增(98℃10秒,66℃30秒,72℃5分钟)。
    31. 根据制造商的说明使用'QIAquick PCR Purification Kit'纯化产品。在23μlEB中洗脱PCR产物。
    32. 为'TCC'和'TCC'的3C控制设置一个对照和三个消化(表1)。在37°C孵育(培养箱,过夜)。

      表1.用于验证末端标记和结扎的消化装置


    33. 加入4μl6x上样染料,在标准2%琼脂糖凝胶上分析产物(Green和Sambrook,2012)。典型结果如图10所示。


      图10.用于标记和结扎末端的典型对照。 两个密切的基因组大麦 Hind III片段在其连接连接处进行PCR扩增。对扩增的3C连接进行III-III消化,其中连接了Hind III的粘端,产生两个不同大小的片段。相反,TCC连接是由填充和标记的 Hind III末端的平末端连接产生的。结果,形成了新的 Nhe I位点并且原始的 Hind III位点丢失(Belton et al。,2012)。通常,约50-80%的PCR产物被 Nh e 切割。表示产品的尺寸(bp)。凝胶图像取自Mascher 等人(2017)。

  10. 从未连接的DNA末端去除生物素和DNA片段化
    1. 向5μgDNA(步骤I28)中加入9μl10xNEBuffer 1和水至84μl。
    2. 加入6μl外切核酸酶III(100U /μl)。
    3. 轻轻混合并孵育(41°C,1小时)(参见注10 )。
    4. 通过添加2μl0.5M EDTA,pH 8.0和2.5μl4M NaCl(Kalhor 等人,2011)来终止反应。
    5. 孵育(70°C,20分钟)并用水调节体积至100μl。
    6. 使用Covaris S220 AFA超声波仪(Duty Factor:10%,PIP:175 W,每次爆发的周期:200,设定模式:频率扫描,时间:60秒)和Covaris microTUBES将DNA剪切至100-500bp。
    7. 通过在标准2%琼脂糖凝胶上运行100ng来控制DNA大小(Green和Sambrook,2012)。如果DNA仍然太大,则添加另一个超声处理循环。
    8. 将样品完全转移到1.5 ml管中。准确确定音量。
    9. 加入1.8体积的AMPure XP珠(室温),混匀,孵育(5分钟)。
    10. 使用MPC回收珠子(2分钟)。
    11. 用190μl70%乙醇洗涤珠子30秒,同时将管子置于MPC中。丢弃上清液。
    12. 重复洗涤步骤(步骤J11)一次。
    13. 完全风干珠子。&nbsp;
    14. 从MPC中取出试管,加入52μlEBT进行洗脱。&nbsp;
    15. 重悬珠子,孵育(1分钟),将管置于MPC中(1分钟)。
    16. 将51μl转移到新管中。
    17. 样品可以在-20°C下储存。

  11. 结束修复和A-tailing
    1. 将加热块设置为20°C。
    2. 对于最终修复,按以下顺序添加碎片,清理的DNA:
      7μl10xTango
      7μl2.5mM dNTP混合物
      0.7μl100mM ATP
      1.5μlT4DNA聚合酶(5 U /μl)
      2.5μlT4多核苷酸激酶(10U /μl)
      0.3μlKlenowDNA聚合酶,大片段(10 U /μl)
      总体积为70μl
    3. 通过上下移液小心混合。
    4. 孵育(20°C,30分钟)样品。
    5. 准确确定音量。
    6. 加入1.8体积的AMPure XP珠(室温),混匀,孵育(5分钟)。
    7. 使用MPC回收珠子(2分钟)。
    8. 用190μl70%乙醇洗涤珠子30秒,同时将管子置于MPC中。丢弃上清液。
    9. 重复洗涤步骤(步骤K8)一次。
    10. 完全风干珠。
    11. 从MPC中取出试管,加入39μlTLE进行洗脱。
    12. 重悬珠子,孵育(1分钟),将管置于MPC中(1分钟)。
    13. 将38μl转移到新管中。
    14. 通过添加以下内容添加“A”:
      5μl10xNEBuffer 2
      4μl2.5mM dATP
      3μlKlenow片段,3'→5'外 - (5U /μl)
      总体积为50μl(Kalhor et al。,2011)
    15. 通过上下移液小心混合并孵育(37°C,30分钟)样品。
    16. 在孵育期间,将加热块设定为65℃。
    17. 通过加入1μl0.5M EDTA,pH 8.0并在65°C温育20分钟来灭活酶。立即将样品放在冰上。
    18. 在灭活期间准备Dynabeads MyOne链霉抗生物素蛋白C1。

  12. 生物素下拉
    1. 所有后续步骤均在1.5ml DNA LoBind管中进行。
    2. Vortex Dynabeads MyOne链霉抗生物素蛋白C1并将10μl转移至1.5ml管中。
    3. 加入400μlTWB,通过移液混合并在摇动平台上孵育珠子3分钟。
    4. 使用MPC回收珠子(1分钟)并弃去上清液。
    5. 从MPC中取出试管,加入400μlTWB并通过移液管混合。
    6. 使用MPC回收珠子(1分钟)并弃去上清液。
    7. 将珠子重悬于50μl2xBB中并加入50μlDNA用于生物素化DNA片段的亲和纯化(Kalhor 等人,,2011)。
    8. 旋转孵育30分钟。
    9. 使用MPC回收珠子(1分钟)并丢弃上清液。
    10. 将珠子重悬于800μl1xBB中,并将悬浮液转移到新管中。
    11. 使用MPC回收珠子(1分钟)并弃去上清液。
    12. 用1x BB重复洗涤步骤。
    13. 从MPC中取出试管,加入100μl1x连接缓冲液并通过移液管混合。
    14. 使用MPC回收珠子(1分钟)并弃去上清液。
    15. 从MPC中取出试管,将珠子重悬于38.8μl1x连接缓冲液中。

  13. Illumina配对末端衔接子连接和PCR
    1. 通过添加1.5毫升管来设置结扎:
      6μlTruSeqDNA单指标(A组)
      38.8μl珠子与亲和纯化的DNA
      1.1μl10xT4 DNA连接缓冲液
      1.1μlPEG4000(50%)
      1.0μl水
      2.0μlT4DNA连接酶(5 U /μl)。
      注意:要选择“TruSeq DNA索引”,请按照“TruSeq Library Pooling Guide”(Illumina,San Diego,California,U.S.A。)中提供的说明进行操作。
      通过移液充分混合并孵育1小时(22°)。
    2. 使用MPC回收珠子(1分钟)并弃去上清液。
    3. 将珠子重悬于400μlTWB中并旋转5分钟。
    4. 使用MPC回收珠子(1分钟)并弃去上清液。
    5. 用400μlTWB重复洗涤两次。
    6. 在200μl1xBB中重悬珠子。
    7. 使用MPC回收珠子(1分钟)并弃去上清液。
    8. 在200μl1xNEBuffer 2中重悬珠子。
    9. 使用MPC回收珠子(1分钟)并弃去上清液。
    10. 用200μl1xNEBuffer 2重复洗涤。
    11. 从MPC中取出管子。
    12. 将珠子重悬于20μl1xNEBuffer 2中。
    13. 将样品转移到新的1.5ml管中并将珠子结合的TCC DNA储存在冰上。
    14. 滴定最佳PCR循环次数(Belton et al。,2012)。
      重悬珠子并加入PCR管:
      1.5μl珠子结合的TCC DNA
      1.3μl10μM引物3 + 4(参见注11 )
      5.0μl5xQ5热启动缓冲区
      2.0μl2.5mM dNTP混合物
      0.3μlQ5热启动高保真DNA聚合酶(2 U /μl)
      14.9μl水(终体积25μl)。
    15. 滴定PCR:在98°C(30秒)初始培养后,进行DNA扩增(20个循环:98°C,10秒,68°C,30秒,72°C,30秒),然后在72°C下最后延伸5分钟。
    16. 在9,12,15和20个循环后取4μl等分试样。
    17. 使用标准琼脂糖凝胶分析等分试样(图11)。通过与来自梯子的已知DNA量进行比较来估计产品的量。选择PCR循环次数和反应次数(25μl体积)以产生约200-600ng DNA用于测序(参见注释12 )。为了避免PCR产物的大小分布变化所指示的PCR伪影,建议使用最多15个循环(Belton 等人,,2012)。图11所示的文库在12个循环中产生了足够量的DNA。


      图11.滴定PCR。在指定数量的PCR循环后,取出2.4μl反应(初始体积的PCR:25μl),使用标准2%琼脂糖凝胶进行大小分离并染色与溴化乙锭。表示GeneRuler 50bp DNA梯(L)的大小(bp)。 500 bp标记条带对应20 ng DNA。

    18. 重悬珠结合的TCC DNA并用计算的反应数设置最终的PCR。使用最佳循环次数进行PCR。

  14. 反应清理和尺寸选择
    1. 将所有PCR产物汇集在一个1.5ml管中,并将管置于MPC中。
    2. 等待1分钟,将含有文库的上清液转移到新的1.5 ml管中进行清理和大小选择(Belton et al。,2012)。
    3. 准确确定音量。
    4. 加入1.8体积的AMPure XP珠粒(室温),混合,孵育(5分钟)并用MPC回收珠粒(2分钟)。
    5. 用190μl70%乙醇洗涤珠子30秒,同时将管子置于MPC中。
    6. 丢弃上清液。
    7. 重复洗涤步骤(步骤N5-N6)一次。
    8. 完全风干珠。
    9. 从MPC中取出试管,加入27μlEBT进行洗脱。
    10. 重悬珠子并孵育(10分钟)。每2分钟轻拍一次试管,重新悬浮珠子。
    11. 将管置于MPC中(1分钟)并将25μl转移至新管中。
    12. 使用Qubit 2.0(或Qubit 4)荧光计和dsDNA BR Assay确定DNA浓度。浓度应为10-30 ng /μl。
    13. 使用凝胶纯化确保使用适当大小的DNA片段进行测序(Himmelbach et al。,2014)(参见注释13 )。在500毫升Erlenmeyer烧瓶中称取1克UltraPure琼脂糖。加入50ml 1x TAE缓冲液,用微波炉融化琼脂糖直至完全溶解。将液体冷却至60°C,加入5μlSYBR-Gold(参见注释14 ),轻轻混合,不产生气泡,并使用标准电泳设备浇注凝胶。用铝箔覆盖凝胶,让凝胶固化(光敏染料)。
    14. 将250 ng GeneRuler 50 bp DNA ladder转移到试管中,加入4μl6x上样染料,并使用EBT将体积调节至24μl。通过添加5μl6x上样染料制备TCC样品(25μl)。
    15. 取出梳子,将凝胶放入装有1x TAE缓冲液的电泳室中。将TCC库加载到两个相邻的插槽中。将标准物应用于一个泳道,以5 V / cm(阳极和阴极之间的距离)进行电泳1小时。在分离过程中保护凝胶免受光照。使用“暗读者”透照仪(参见注释15 )可视化DNA并拍照。使用干净的手术刀切除300到550 bp之间的区域(图12)。


      图12.典型TCC文库的凝胶纯化。 在大小分离(标准2%琼脂糖凝胶电泳)和用SYBR Gold染色后,使用可见蓝光发射的“暗读取器透照仪”显示该文库。提取来自约300至550bp的框架区域的DNA用于测序。表示GeneRuler 50 bp DNA ladder(L)的大小(bp)。

    16. 将凝胶块放入2.0 ml管中,用天平确定体积(例如,234 mg的块对应234μl)。
    17. 使用'MinElute Gel Extraction Kit'纯化DNA,基本上如制造商所述。加入6倍体积的QG缓冲液并在温和搅拌下完全溶解凝胶。
    18. 加入一个凝胶体积的异丙醇并通过倒置混合。
    19. 将700μl溶解的凝胶应用于MinElute柱。将柱置于收集管中并旋转(16,000 x g ,1分钟)。丢弃流通。
    20. 逐步将残余液体添加到柱中(重复步骤N19)。
    21. 加入740μlPE洗涤柱,孵育3分钟,旋转柱(16,000 x g ,1分钟)并丢弃流通液(参见注释16 )。
    22. 转动转子中的色谱柱(180°)并旋转(16,000 x g ,1分钟)。
    23. 丢弃收集管,将MinElute柱置于干净的1.5 ml管中,并进行洗脱,向树脂中加入50μlEB。
    24. 孵育1分钟并旋转柱(16,000 x g ,1分钟)。
    25. 将洗脱的TCC文库保存在-20°C。

  15. 质量控制,量化和测序
    1. 使用Qubit 2.0(或Qubit 4)荧光计(ds DNA HS Assay)测量DNA浓度。浓度应为10-20 ng /μl。
    2. 用 Nhe I消化80ng TCC文库以估计来自生物素化连接的连接的部分(Belton 等人,,2012)。通过添加到1.5 ml管中来设置反应:
      80 ng TCC库
      2μl10xTango缓冲液
      1μl Nhe I(10 U /μl)
      水最终体积为20μl
      准备一个未切割的对照(没有 Nhe 我消化)。在37°C孵育过夜。使用2μlGeneRuler50bp DNA ladder作为标准,用2%琼脂糖凝胶分析产物。消化的TCC文库的较小尺寸分布提供了含有真正TCC连接产物的分数的估计值(图13)。


      图13.最终TCC文库的质量控制。用 Nhe 消化后,将TCC文库(80 ng)进行大小分离(标准2%琼脂糖凝胶),染色与溴化乙锭相比,未切割的对照组(80 ng)。向较小尺寸分布的转变表明存在 Nhe I位点,这些位点源自真正的TCC结扎事件(Belton et al。,2012; Mascher 等人。,2017)。总的来说,我们发现这种转变是一种安全的质量指标。没有明确转变的文库不应该被测序,因为它们仅产生非常小部分有用的TCC读数。表示GeneRuler 50 bp DNA ladder(L)的大小(bp)。

    3. 记录文库的大小配置文件,并使用Agilent高灵敏度DNA试剂盒和Agilent 2100生物分析仪确定TCC文库的平均大小(图14)。


      图14.典型TCC文库的大小分布。使用Agilent 2100生物分析仪对TCC文库进行大小分级。平均文库大小(区域1:434bp;包括121bp Illumina P5和P7衔接子)对应于约313bp的平均插入片段大小。 LM:较低的标记峰。 UM:上标记峰。 FU:荧光单位。 bp:碱基对。

    4. 使用所描述的Illumina系统(Mascher et al。,2013)对文库进行定量和测序(配对末端,2x100个循环)。

数据分析

主要序列数据分析

  1. 运行Illumina CASAVA管道( http://support.illumina.com/sequencing/sequencing_software/casava .html )以FASTQ格式获取解卷积的读取文件。
  2. 使用适配器序列'AAGCTAGCTT'修剪cutadapt(Martin,2011)在连接位点的读数。
  3. 使用BWA mem将修剪的读取对与适当的参考基因组对齐(Li,2013)。通过指定参数'-S'和'-P',应将两个读数映射为单端。参数'-M'应该用于将较短的分割命中标记为次要命名。
  4. 按引用位置对BAM文件进行排序以删除重复项。然后通过读取名称再次对BAM文件进行排序,以使一对的两端在BAM文件的相邻行中。其中一个工具SAMtools(Li et al。,2009),Picard( http:/ /broadinstitute.github.io/picard/ )或Novosort( http://www.novocraft .com / products / novosort / )可以使用。
  5. 使用非唯一对齐删除读取,并使用命令'samtools view -q 10 -F1284'丢弃二次比对,未映射的读取和重复读取。
  6. 使用BEDTools为限制片段分配读数(Quinlan和Hall,2010)。您将需要一个BED文件( https://genome.ucsc.edu/FAQ/FAQformat。 html#format1 )从 Hind III获得的限制性片段(即,两个 Hind III位点之间的区域)的位置 in silico 您的参考基因组序列的消化。使用命令'bedtools pairtobed -bedpe -f 1 -type both -abam'。生成的BEDPE文件是一个制表符分隔文件,可以使用标准的UNIX文本处理工具(如AWK或Perl)进行处理。一对的两端到限制片段的分配在相邻的线上。
  7. 使用UNIX脚本(例如。,在AWK语言中)根据对齐起始位置到相邻 Hind III站点的距离来计算插入大小。丢弃插入物大小超过500 bp的片段,绘制插入物大小分布(图15)并将其与使用Agilent Bioanalyzer获得的片段进行比较。


    图15. TCC文库的典型计算机尺寸分布。将序列数据映射到参考基因组并分配给限制性片段。每个测序片段的插入大小由比对起始位置与下一个 Hind III限制位点的距离确定。分布模式用红线表示。

笔记

  1. 联系当地销售代表,了解订购,性能和配件。
  2. 如果未观察到叶子变黑,则固定剂可能未完全进入,因此表明交联不完全。优化渗透条件,例如通过增加真空或切割较短的植物区段。
  3. 必须穿戴适当的防护装备。请咨询贵机构的安全官员,以正确处理液氮。
  4. MyOne链霉抗生物素蛋白T1珠子每毫升提供约250cm 2 表面。通过使用400μl珠子,纯化的染色质以低密度束缚在约100cm 2 的大表面上(Kalhor 等人,2011)。因此有利于DNA末端的分子内连接。
  5. 游离链霉抗生物素蛋白残基被生物素饱和,以避免干扰生物素-14-dCTP,其用于在随后的步骤中标记DNA末端(Kalhor 等人,2011)。
  6. 在该反应过程中,产生钝性DNA末端,其被生物素化的胞嘧啶残基标记,所述生物素化的胞嘧啶残基位于通过掺入dGTPαS引入的硫代磷酸酯键的3'处(Kalhor 等人,2011)。
  7. 在平衡至室温的离心机中旋转样品。低温可能导致形成混浊的水相。在转移过程中避免用间期的蛋白质污染水相。
  8. NaCl用于DNA沉淀,因为该溶液含有SDS(Green和Sambrook,2012)。随后,在乙酸钠存在下沉淀DNA以除去痕量的氯化钠,这对外切核酸酶III具有抑制作用(Hoheisel,1993)。
  9. 连接的 Hind III位点(AAGCTT)的连接产生限制酶 Nhe I(GCTAGC)的位点。为了控制填充和平末端连接的有效性,产生来自两个相邻大麦 Hind III /限制片段的PCR片段(534bp)。为了扩增,使用引物1和引物2。
  10. 来自 E的外切核酸酶III。大肠杆菌催化从双链DNA的3'-羟基末端逐步除去单核苷酸(New England BioLabs; Rogers and Weiss,1980)。因此,外切核酸酶III允许从未连接的DNA末端去除生物素化的残基。因为位于生物素化胞嘧啶残基5'的硫代磷酸酯键不被外切核酸酶III切割(Putney et al。,1981),真正的连接连接被保留(Kalhor et al。 ,2011)。
  11. 引物3和4与Illumina适配器退火并允许扩增文库。
  12. 在常规实验中,11-12个扩增循环就足够了。 8个PCR反应(每个反应25μl体积)应产生足够的DNA用于测序。遵循“黄金法则”以维持文库的复杂性:保持较低的循环次数并增加PCR反应的数量以获得足够的测序文库(Belton et al。,2012)。
  13. Sage Science的BluePippin是标准凝胶纯化的替代品。
  14. 不要使用溴化乙锭染色的凝胶。用于激发溴化乙锭的紫外线辐射会破坏DNA。
  15. 使用SYBR-Gold染料在琼脂糖凝胶中显示DNA,并从'暗读者'透射仪发射的可见蓝光作为激发源。
  16. 将色谱柱始终放置在转子中,因为色谱柱将旋转180°以去除液体痕迹。

食谱

注意:

  1. 所有溶液均使用纯净水(GenPure Pro UV / UF)制备,并在室温下储存,除非另有说明。
  2. 在整个实验过程中必须采用良好的实验室规范。
  3. 咨询您所在机构的安全数据表和实验室/环境安全规则以正确处理试剂和仪器尤为重要。

  1. 1 M HEPES,pH 8.0(100 ml)
    1. 称取26.0克HEPES钠盐(4-(2-羟乙基)哌嗪-1-乙磺酸钠盐)并加入70毫升水
    2. 用pH计用浓盐酸搅拌并调节pH至8.0。用水调至100毫升
    3. 使用0.2μm过滤器和50 ml注射器灭菌(在室温下在黑暗中储存溶液)
  2. 2 M蔗糖(40 ml)
    1. 将27.4g蔗糖装入50ml管中
    2. 加水至38毫升,将50毫升管置于温水浴中。
    3. 以规则的间隔涡旋温热的混合物以完全溶解蔗糖
    4. 将体积调节至40 ml并将溶液储存在-20°C
    5. 使用前在温水浴中解冻
  3. 1 M KCl(100 ml)
    1. 将7.5克KCl溶于水中至终体积为100毫升
    2. 使用量筒调节体积并通过高压灭菌消毒
    3. 在室温下储存
  4. 10%(v / v)Triton X-100(45 ml)
    1. 将5ml Triton X-100加入45ml水中,加入50ml管中
    2. 轻轻混合,避免起泡
    3. 储存10%Triton X-100和含有Triton X-100的溶液,在室温下避光保存
  5. 含甲醛的细胞核分离缓冲液(NIBF)(Hövel et al。,2012)(200 ml)
    将所有组分加入250毫升瓶中,并在使用前储存在冰上:
    4毫升1M HEPES,pH 8.0
    25毫升2 M蔗糖
    200μl1MMgCl 2
    1毫升1M KCl
    100.8g 100%(v / v)甘油(相当于80ml)
    5毫升10%(v / v)Triton X-100
    10.7毫升37%(w / v)甲醛
    200μl100mM PMSF
    0.1%(v / v)2-巯基乙醇
    加水至最终体积为200毫升
    注意:PMSF抑制丝氨酸蛋白酶,在水中不稳定。由于pH值为8.0时的半衰期为35分钟(James,1978),因此必须在使用前立即加入PMSF。在空气中,甲醛慢慢氧化成甲酸。购买少量并在购买后立即使用。质量差的甲醛会破坏实验。在室温下储存甲醛,因为在寒冷中可能形成沉淀物。在使用前,在通风橱内加入甲醛,PMSF和2-巯基乙醇。彻底搅拌并将溶液冷却在冰上。在组合物之后立即使用NIBF进行真空渗透。 100%甘油(密度1.26g / ml)是粘性的并粘在移液管和量筒上。为方便起见,在玻璃瓶中称重100.8g 100%甘油,相当于80ml。
  6. 2 M甘氨酸(40 ml)
    1. 称取6.0g甘氨酸加入50ml管中,用水调至40ml
    2. 储存于-20°C并在使用前在温水浴中解冻
  7. 1 mg / ml抑肽酶(Haring et al。,2007)
    1. 将1毫克Apronitin溶于1毫升水中
    2. 以-20°C的等分试样储存&nbsp;
  8. 1 mg / ml Leupeptin(Haring et al。,2007)
    1. 将1mg Leupeptin溶于1ml水中
    2. 以-20°C的等分试样储存&nbsp;
  9. 1 mg / ml Pepstatin(Haring et al。,2007)
    1. 将1mg Pepstatin溶于1ml水中
    2. 以-20°C的等分试样储存&nbsp;
  10. 具有蛋白酶抑制剂的核分离缓冲液(NIBP)(Hövel et al。,2012)(150 ml)
    将所有组分加入250毫升瓶中,并在使用前储存在冰上:
    3毫升1M HEPES,pH 8.0
    18.8毫升2 M蔗糖
    150μl1MMgCl 2
    750μl1M KCl
    75.6g 100%(v / v)甘油(相当于60ml)
    3.8毫升10%(v / v)Triton X-100
    150μl100mM PMSF
    150μl100%(v / v)2-巯基乙醇
    150μl1mg / ml抑肽酶
    150μl1mg / ml Leupeptin
    150μl1mg / ml Pepstatin
    加水至最终体积150毫升
    注意:PMSF和2-巯基乙醇在使用前加入通风橱内。彻底搅拌并将溶液冷却在冰上。组成后立即使用NIBP。 100%甘油(密度1.26g / ml)是粘性的并粘在移液管和量筒上。为方便起见,在玻璃瓶中称重75.6克100%甘油,相当于60毫升。
  11. 蔗糖垫(20毫升)
    将所有组分加入50毫升塑料管中,并在使用前储存在冰上:
    400μl1M HEPES,pH 8.0
    16.9毫升2 M蔗糖
    20μl1MMgCl 2
    100μl1M KCl
    500μl10%(v / v)Triton X-100
    20μl100mM PMSF
    20μl100%(v / v)2-巯基乙醇
    20μl1mg / ml抑肽酶
    20μl1mg / ml Leupeptin
    20μl1mg / ml Pepstatin
    加水至最终体积为20毫升
    注意:PMSF和2-巯基乙醇在使用前加入通风橱内。彻底搅拌并将溶液冷却在冰上。成分后立即使用蔗糖垫。
  12. 细胞核分离缓冲液,含1.5 M蔗糖(NIBS)(20 ml)
    将所有组分加入50毫升塑料管中,并在使用前储存在冰上:
    400μl1M HEPES,pH 8.0
    14.9毫升2 M蔗糖
    20μl1MMgCl 2
    100μl1M KCl
    500μl10%(v / v)Triton X-100
    20μl100mM PMSF
    20μl100%(v / v)2-巯基乙醇
    20μl1mg / ml抑肽酶
    20μl1mg / ml Leupeptin
    20μl1mg / ml Pepstatin
    加水至最终体积为20毫升
    注意:PMSF和2-巯基乙醇在使用前加入通风橱内。彻底搅拌并将溶液冷却在冰上。合成后立即使用NIBS。
  13. 2%(w / v)SDS(100ml)
    1. 将2g SDS(十二烷基硫酸钠)溶于70ml水中
    2. 轻轻混合,避免起泡,并使用量筒将体积调节至100毫升
    3. 溶液可以在室温下储存
  14. 4 M NaCl(100 ml)
    1. 将23.34g NaCl溶于80ml水中
    2. 使用量筒将体积调节至100 ml,通过高压灭菌消毒并在室温下储存
  15. 洗涤缓冲液1(50毫升)
    将所有组分加入50毫升塑料管中:
    2.5ml 1M Mris-HCl,pH 8.0
    625μl4M NaCl
    100μl0.5MEDTA,pH 8.0
    用水调节至最终体积为50 ml(Kalhor et al。,2011)
  16. 25 mM IPB(150μl)
    将2 mg EZlink Iodoacetyl-PEG 2 -Biotin(IPB)溶于洗涤缓冲液1(最终体积150μl)
    注意:使用前请准备好新溶液。 IPB对水分和光敏感。将试剂储存在4-8°C干燥的密闭暗容器中,同时加入干燥的硅酸盐珠粒。在打开之前,将样品瓶平衡至室温,以防止样品瓶中的水分凝结。
  17. 10x NEBuffer 2(New England BioLabs)(10 ml)
    添加所有组件:
    1.25毫升4 M NaCl
    1毫升1M Tris-HCl,pH 8.0
    1毫升1M MgCl 2
    100μl1M DTT
    加水至10毫升,并以-20℃的等分试样储存
  18. 1x NEBuffer 2
    通过用9ml水稀释1ml 10x NEBuffer 2原液来制备
    将等分试样储存在-20°C
  19. Hind III(10 U /μl)
    将所有组分加入冰上并轻轻混合:
    0.5μl Hind III / III(100U /μl)
    4.5μl1xNEBuffer 2
    使用前新鲜准备
  20. 透析缓冲液(TE缓冲液)(2 L)
    添加所有组件:
    20ml 1M Mris-HCl,pH 8.0
    4 ml 0.5 M EDTA,pH 8.0
    加水至最终体积为2升
  21. 磷酸盐缓冲盐水与吐温20,pH 7.4(PBST)(1升)
    添加所有组件:
    8.0克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24克KH 2 PO 4
    加水至800毫升
    使用HCl将pH调节至7.4(Green and Sambrook,2012)
    加入1ml 10%Tween 20和水至终体积为1L
  22. 25 mM中和的IPB(75μl)
    1. 将1 mg EZlink Iodoacetyl-PEG 2 -Biotin(IPB)溶于洗涤缓冲液1(最终体积75μl)
    2. 取40μl25mMIPB并加入7μl143mM2-巯基乙醇
    注意:使用前请准备好新的解决方案。
  23. 10 mM dATP
    用900μl水稀释100μl100mM dATP并储存在-20°C
  24. 10 mM dTTP
    用900μl水稀释100μl100mM dTTP并储存在-20°C
  25. 洗涤缓冲液2(50ml)(Kalhor et al。,2011)
    将所有组分加入50毫升塑料管中:
    2.5ml 1M Mris-HCl,pH 8.0
    625μl4M NaCl
    2毫升10%(v / v)Triton X-100
    用水调节至最终体积为50毫升&nbsp;
  26. 洗涤缓冲液3(50ml)(Kalhor et al。,2011)
    将所有组分加入50毫升塑料管中:
    2.5毫升1M Tris-HCl,pH 7.4
    10μl0.5MEDTA,pH 8.0
    2毫升10%(v / v)Triton X-100
    用水调节至最终体积为50毫升&nbsp;
  27. 10x连接缓冲液TCC(1.5 ml)
    将所有组件加入2.0毫升塑料管中:
    750μl1MTris-HCl,pH 7.5
    150μl1MMgCl 2
    150μl100mM ATP
    150μl1M DTT
    300μl水
    注意:&nbsp;
    1. 使用前新鲜配制溶液。
    2. 该组合物与来自New England BioLabs的10x反应缓冲液相同。
  28. 10毫克/毫升BSA&nbsp;
    1. 将200μlUltrapureBSA(50mg / ml)转移到管中
    2. 加入800μl水,混合并储存在-20°C
  29. 提取缓冲液(50 ml)(Kalhor et al。,2011)
    添加所有组件:
    2.5ml 1M Mris-HCl,pH 8.0
    100μl0.5MEDTA,pH 8.0
    1.25毫升4 M NaCl
    1毫升10%(v / v)SDS
    用水调节至最终体积为50毫升&nbsp;
  30. 25 mg / ml RNase A(1 ml)
    将25 mg RNase A(不含DNA酶)溶于1 ml终体积的水中,并在4°C下储存
  31. 20 mg / ml蛋白酶K(150μl)
    将3mg蛋白酶K溶于150μl10mMTris-HCl,pH 8.0中 在使用前刚刚准备好解决方案
  32. 5 mg / ml糖原(100μl)
    用75μl水稀释25μl糖原(20mg / ml)并储存在-20℃
  33. 80%乙醇(100毫升)
    混合80毫升100%乙醇和20毫升水
    准备新鲜并存放在一个紧闭的瓶子里
    使用前在冰上预先冷却。
  34. 70%乙醇(100毫升)
    混合70毫升100%乙醇和30毫升水
    准备新鲜并存放在一个紧闭的瓶子里
    使用前在冰上预先冷却。
  35. 3 M乙酸钠,pH 5.2(100 ml)
    如Green和Sambrook(2012)所述制备100ml溶液。
  36. EB(50毫升)
    添加所有组件:
    500μl1MTris-HCl,pH 8.0
    49.5毫升水
  37. EBT(50毫升)
    添加所有组件:
    500μl1MTris-HCl,pH 8.0
    250μl10%Tween 20
    49.25毫升水
  38. 引物1 + 2(各10μM)(100μl)
    引物1(100μM,5'-ATCTTCATGCGAGGCAGAGT -3')
    引物2(100μM,5'-ACCGTTGAACCATCTTCAGG -3')
    添加所有组件:
    10μl100μM引物1(100μM)
    10μl100μM引物2(100μM)
    80μl水
    在-20°C下储存大麦的引物混合物(生物素掺入对照)(Mascher et al。,2017)
  39. 10 mM dNTP混合液(1 ml)
    将400μldNTP混合物(各25mM)加入600μl水中并储存在-20°C
  40. 2.5 mM dNTP混合液(1 ml)
    将100μldNTP混合物(各25mM)加入900μl水中并在-20℃下储存
  41. TLE(50 ml)
  42. Tween Wash Buffer(TWB)(50 ml)(Lieberman-Aiden et al。,2009)
    添加所有组件:
    250μl1MTris-HCl,pH 8.0
    50μl0.5MEDTA,pH 8.0
    250μl10%Tween 20
    12.5毫升4 M NaCl
    用水调节至最终体积为50毫升&nbsp;
  43. 2x结合缓冲液(2x BB)(50 ml)(Lieberman-Aiden et al。,2009)
    添加所有组件:
    500μl1MTris-HCl,pH 8.0
    100μl0.5MEDTA,pH 8.0
    25毫升4 M NaCl
    用水调节至最终体积为50毫升
  44. 1x结合缓冲液(1x BB)(20 ml)(Lieberman-Aiden et al。,2009)
    用10毫升水稀释10毫升2倍BB&nbsp;
  45. 1x连接缓冲液(1 ml)
    添加所有组件:
    90μl10xT4 DNA连接缓冲液
    90μl50%PEG 4000
    820μl水
    储存在-20°C
  46. 引物3 + 4(各10μM)(100μl)
    引物3(100μM,5'-AATGATACGGCGACCACCGAGAT-3')
    引物4(100μM,5'-CAAGCAGAAGACGGCATACGA-3')
    添加所有组件:
    10μl100μM引物3(100μM)
    10μl100μM引物4(100μM)
    80μl水
    将Illumina文库扩增引物混合物储存在-20°C
  47. 6x上样染料(1毫升)
    添加所有组件:
    700μl水
    300μl100%(v / v)甘油
    一点点溴酚蓝
    注意:添加溴酚蓝以获得浅蓝色。超标将在标准琼脂糖凝胶中掩盖DNA(面积在200-300bp之间)。
  48. 50x TAE(1升)(Green and Sambrook,2012)
    1. 将242克Tris碱溶于去离子水中
    2. 加入57.1ml冰醋酸和100ml 0.5M EDTA,pH8.0
    3. 将音量调节至1 L并充分混合
    4. 将50x TAE原液用水稀释至1x TAE工作溶液并充分混合

致谢

这项工作得到了莱布尼茨植物遗传和作物植物研究所(IPK),Gatersleben的核心资金以及德国联邦教育和研究部(BMBF,FKZ 0315954)向Nils的项目资金('TRITEX')的资助。斯坦。感谢Karin Lipfert的平面设计。作者感谢Lala Aliyeva-Schnorr对细胞核的微观分析提供帮助。该方案在很大程度上适用于湿实验室程序(Hövel等人,2012; Lieberman-Aiden等人,2009; Kalhor等人,2011; Belton等人,2012)和先前描述的生物信息学程序(Mascher等人,2017; Beier等人。 ,2017)。作者声明没有竞争利益或利益冲突。

参考

  1. Abdalla,K.O.,Thomson,J。A.和Rafudeen,M。S.(2009)。 从复活植物Xerophyta viscosa进行核分离和核蛋白提取的蛋白质组学研究协议。 Anal Biochem 384(2):365-367。
  2. Avni,R.,Nave,M.,Barad,O.,Baruch,K.,Twardziok,SO,Gundlach,H.,Hale,I.,Mascher,M.,Spannagl,M.,Wiebe,K.,Jordan ,KW,Golan,G.,Deek,J.,Ben-Zvi,B.,Ben-Zvi,G.,Himmelbach,A.,MacLachlan,RP,Sharpe,AG,Fritz,A.,Ben-David,R 。,Budak,H.,Fahima,T.,Korol,A.,Faris,JD,Hernandez,A.,Mikel,MA,Levy,AA,Steffenson,B.,Maccaferri,M.,Tuberosa,R.,Cattivelli ,L.,Faccioli,P.,Ceriotti,A.,Kashkush,K.,Pourkheirandish,M.,Komatsuda,T.,Eilam,T.,Sela,H.,Sharon,A.,Ohad,N.,Chamovitz ,DA,Mayer,KFX,Stein,N.,Ronen,G.,Peleg,Z.,Pozniak,CJ,Akhunov,ED和Distelfeld,A。(2017)。 野生二粒基因组结构和多样性阐明了小麦的进化和驯化。 科学< / em> 357(6346):93-97。
  3. Beier,S.,Himmelbach,A.,Colmsee,C.,Zhang,XQ,Barrero,RA,Zhang,Q.,Li,L.,Bayer,M.,Bolser,D.,Taudien,S.,Groth, M.,Felder,M.,Hastie,A.,Simkova,H.,Stankova,H.,Vrana,J.,Chan,S.,Munoz-Amatriain,M.,Ounit,R.,Wanamaker,S。, Schmutzer,T.,Aliyeva-Schnorr,L.,Grasso,S.,Tanskanen,J.,Sampath,D.,Heavens,D.,Cao,S.,Chapman,B.,Dai,F.,Han,Y 。,Li,H.,Li,X.,Lin,C.,McCooke,JK,Tan,C.,Wang,S.,Yin,S.,Zhou,G.,Poland,JA,Bellgard,MI,Houben ,A.,Dolezel,J.,Ayling,S.,Lonardi,S.,Langridge,P.,Muehlbauer,GJ,Kersey,P.,Clark,MD,Caccamo,M.,Schulman,AH,Platzer,M。 ,Close,TJ,Hansson,M.,Zhang,G.,Braumann,I.,Li,C.,Waugh,R.,Scholz,U.,Stein,N。和Mascher,M。(2017)。 构建基于地图的大麦参考基因组序列 Hordeum vulgare L. Sci Data 4:170044。
  4. Belton,J.M.,McCord,R.P.,Gibcus,J.H.,Naumova,N.,Zhan,Y。和Dekker,J。(2012)。 Hi-C:捕获基因组构象的综合技术。 方法 58(3):268-276。
  5. Burton,J.N.,Adey,A.,Patwardhan,R.P.,Qiu,R.,Kitzman,J.O。和Shendure,J。(2013)。 基于染色质相互作用的从头基因组装配的染色体尺度支架。 Nat Biotechnol 31(12):1119-1125。
  6. Dekker,J.,Rippe,K.,Dekker,M。和Kleckner,N。(2002)。 捕获染色体构象。 Science 295(5558): 1306-1311。
  7. de Wit,E。和de Laat,W。(2012)。 三十年的3C技术:洞察核组织。 Genes Dev < / em> 26(1):11-24。
  8. Duan,Z.,Andronescu,M.,Schutz,K.,McIlwain,S.,Kim,Y。J.,Lee,C.,Shendure,J.,Fields,S.,Blau,C.A。and Noble,W。S.(2010)。 酵母基因组的三维模型。 Nature 465(7296):363-367。
  9. Dudchenko,O.,Batra,S.S.,Omer,A.D.,Nyquist,S.K.,Hoeger,M.,Durand,N.C.,Shamim,M.S.,Machol,I.,Lander,E.S.,Aiden,A.P。和Aiden,E.L。(2017)。 使用Hi-C重新组装埃及伊蚊基因组,产生染色体长度的支架。 Science 356(6333):92-95。
  10. Feng,S.,Cokus,S.J。,Schubert,V.,Zhai,J.,Pellegrini,M。和Jacobsen,S.E。(2014)。 野生型和突变体中的全基因组Hi-C分析显示拟南芥中的高分辨率染色质相互作用。 Mol Cell 55(5):694-707。
  11. Green,M。和Sambrook,J。(2012)。 分子克隆。实验室手册。纽约冷泉港实验室。
  12. Grob,S.,Schmid,M。W.和Grossniklaus,U。(2014)。 拟南芥中的Hi-C分析识别KNOT,这是一种具有相似性的结构到 Drosophila 的弗拉门戈基因座。 Mol Cell 55(5):678-693。
  13. Haring,M.,Offermann,S.,Danker,T.,Horst,I.,Peterhansel,C。和Stam,M。(2007)。 染色质免疫沉淀:优化,定量分析和数据标准化。 植物方法< / em> 3:11。
  14. Himmelbach,A.,Knauft,M。和Stein,N。(2014)。 针对Illumina测序优化的植物序列捕获。 Bio-protocol 4(13): 。e1166&NBSP;
  15. Hoheisel,J。D.(1993)。 关于大肠杆菌外切核酸酶III的活性。 Anal Biochem 209(2):238-246。
  16. Hövel,I.,Louwers,M。和Stam,M。(2012)。 植物中的3C技术。 方法 58(3) :204-211。
  17. James,G。T.(1978)。 在缓冲液中灭活蛋白酶抑制剂苯甲基磺酰氟。 Anal Biochem 86(2):574-579。
  18. Kalhor,R.,Tjong,H.,Jayathilaka,N.,Alber,F。和Chen,L。(2011)。 通过系留染色体构象捕获和基于群体的建模揭示的基因组架构。 Nat Biotechnol 30(1):90-98。
  19. Kaplan,N。和Dekker,J。(2013)。 体内 DNA相互作用频率的高通量基因组支架。 Nat Biotechnol 31(12):1143-1147。
  20. Li,H。(2013)。&nbsp; 使用BWA-MEM对齐序列读数,克隆序列和装配重叠群。 arXiv preprint arXiV:1303.3997。
  21. Li,H.,Handsaker,B.,Wysoker,A.,Fennell,T.,Ruan,J.,Homer,N.,Marth,G.,Abecasis,G.,Durbin,R。和Genome Project Data Processing, S.(2009)。 序列比对/图谱格式和SAMtools。 生物信息学 25(16):2078-2079。
  22. Lieberman-Aiden,E.,van Berkum,NL,Williams,L.,Imakaev,M.,Ragoczy,T.,Telling,A.,Amit,I.,Lajoie,BR,Sabo,PJ,Dorschner,MO,Sandstrom ,R.,Bernstein,B.,Bender,MA,Groudine,M.,Gnirke,A.,Stamatoyannopoulos,J.,Mirny,LA,Lander,ES和Dekker,J。(2009)。 远程互动的综合映射揭示了人类基因组的折叠原则。 科学 326(5950):289-293。
  23. Liu,C.,Wang,C.,Wang,G.,Becker,C.,Zaidem,M。和Weigel,D。(2016)。 单基因拟南芥中染色质包装的全基因组分析分辨率。 Genome Res 26(8):1057-1068。
  24. Martin,M。(2011)。 Cutadapt从高通量测序读数中删除适配器序列。 EMBnet.journal v.17,n。 1,p。第10-12页,可能。 ISSN 2226-6089。
  25. Mascher,M.,Richmond,TA,Gerhardt,DJ,Himmelbach,A.,Clissold,L.,Sampath,D.,Ayling,S.,Steuernagel,B.,Pfeifer,M.,D'Ascenzo,M。, Akhunov,ED,Hedley,PE,Gonzales,AM,Morrell,PL,Kilian,B.,Blattner,FR,Scholz,U.,Mayer,KF,Flavell,AJ,Muehlbauer,GJ,Waugh,R.,Jeddeloh,JA和Stein,N。(2013)。 大麦全外显子组捕获:大麦属及其他基因组研究的工具。 Plant J 76(3):494-505。
  26. Mascher,M.,Gundlach,H.,Himmelbach,A.,Beier,S.,Twardziok,SO,Wicker,T.,Radchuk,V.,Dockter,C.,Hedley,PE,Russell,J.,Bayer, M.,Ramsay,L.,Liu,H.,Haberer,G.,Zhang,XQ,Zhang,Q.,Barrero,RA,Li,L.,Taudien,S.,Groth,M.,Felder,M。 ,Hastie,A.,Simkova,H.,Stankova,H.,Vrana,J.,Chan,S.,Munoz-Amatriain,M.,Ounit,R.,Wanamaker,S.,Bolser,D.,Colmsee, C.,Schmutzer,T.,Aliyeva-Schnorr,L.,Grasso,S.,Tanskanen,J.,Chailyan,A.,Sampath,D.,Heavens,D.,Clissold,L.,Cao,S。, Chapman,B.,Dai,F.,Han,Y.,Li,H.,Li,X.,Lin,C.,McCooke,JK,Tan,C.,Wang,P.,Wang,S.,Yin ,S.,Zhou,G.,Poland,JA,Bellgard,MI,Borisjuk,L.,Houben,A.,Dolezel,J.,Ayling,S.,Lonardi,S.,Kersey,P.,Langridge,P 。,Muehlbauer,GJ,Clark,MD,Caccamo,M.,Schulman,AH,Mayer,KFX,Platzer,M.,Close,TJ,Scholz,U.,Hansson,M.,Zhang,G.,Braumann,I 。,Spannagl,M.,Li,C.,Waugh,R。和Stein,N。(2017)。 染色体构象捕获大麦基因组的有序序列。 Nature 544(7651):427-433。
  27. Metzker,M。L.(2010)。 测序技术 - 下一代。 Nat Rev Genet 11(1):31-46。
  28. Naumova,N.,Imakaev,M.,Fudenberg,G.,Zhan,Y.,Lajoie,B.R.,Mirny,L。A.和Dekker,J。(2013)。 组织有丝分裂染色体。 Science 342(6161) ):948-953。
  29. Putney,S.D.,Benkovic,S.J。和Schimmel,P.R。(1981)。 一端含有α-硫代磷酸酯核苷酸的DNA片段被不对称地阻断外切核酸酶III的消化和可以在体内复制。 Proc Natl Acad Sci USA 78(12):7350-7354。
  30. Quinlan,A.R。和Hall,I.M。(2010)。 BEDTools:一套灵活的实用工具,用于比较基因组特征。 生物信息学 26(6):841-842。
  31. Rao,S.S.,Huntley,M.H.,Durand,N.C.,Stamenova,E.K.,Bochkov,I.D.,Robinson,J.T。,Sanborn,A.L.,Machol,I.,Omer,A.D.,Lander,E。S.和Aiden,E.L。(2014)。 以千碱基分辨率显示的人类基因组三维图谱揭示了染色质循环的原理。 < em> Cell 159(7):1665-1680。
  32. Rogers,G。和Weiss,B。(1980)。在:Grossman,L。和Moldave,K。(编辑)。 [26] Escherichia coli K-12的核酸外切酶III内切酶。在:酶法中的方法。 65:201-211。纽约:学术出版社。
  33. Sexton,T.,Yaffe,E.,Kenigsberg,E.,Bantignies,F.,Leblanc,B.,Hoichman,M.,Parrinello,H.,Tanay,A。和Cavalli,G。(2012)。 果蝇基因组的三维折叠和功能组织原则。 细胞 148(3):458-472。
  34. Wang,C.,Liu,C.,Roqueiro,D.,Grimm,D.,Schwab,R.,Becker,C.,Lanz,C。和Weigel,D。(2015)。 拟南芥中 Arabidopsis thaliana 的局部染色质包装的全基因组分析。 Genome Res 25(2):246-256。
  35. Zhou,X.,Lowdon,R.F.,Li,D.,Lawson,H.A.,Madden,P.A.,Costello,J.F。和Wang,T。(2013)。 使用WashU Epigenome浏览器探索远程基因组相互作用。 Nat方法 10(5):375-376。
  36. Zimmermann,G.,Baumlein,H.,Mock,H.P.,Himmelbach,A。和Schweizer,P。(2006)。 多基因家族编码大麦的germin样蛋白。基础宿主抗性的调节和功能。 植物生理学 142(1):181-192。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Himmelbach, A., Walde, I., Mascher, M. and Stein, N. (2018). Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly. Bio-protocol 8(15): e2955. DOI: 10.21769/BioProtoc.2955.
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