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Nov 2020
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Simplified Epigenome Profiling Using Antibody-tethered Tagmentation
基于抗体系标记的简化表观基因组图谱   

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Abstract

We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNA in situ. We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.

Keywords: Epigenomic profiling (表观基因组分析), Chromatin accessibility (染色质可及性), RNA polymerase II (RNA聚合酶II), Histone modifications (组蛋白变化), CUT & Tag (目标与标签)

Background

Mapping of DNA accessibility in the chromatin landscape was first described 45 years ago with the observation of DNaseI hypersensitivity at transcriptionally active loci (Weintraub and Groudine, 1976). Because DNaseI preferentially cleaves genomic regions that are depleted of nucleosomes, and regulatory elements are bound by non-histone chromatin proteins rather than nucleosomes, DNaseI hypersensitive site mapping has since been used to characterize the genetic regulatory landscape. Other enzymatic probes of chromatin accessibility include Micrococcal Nuclease (MNase) (Reeves et al., 1978), restriction endonucleases (Jack and Eggert, 1990), transposases (Bownes, 1990), and DNA methyltransferases (Gottschling et al., 1992). Hypersensitive site mapping became more routine with the introduction of genome-wide read-out platforms, beginning with microarrays and later short-read DNA sequencing (Crawford et al., 2004; Dorschner et al., 2004). Chromatin accessibility was also mapped using physical fragmentation and differential recovery of cross-linked chromatin, the basis for FAIRE (Giresi et al., 2007) and Sono-Seq (Auerbach et al., 2009). In recent years, the most popular chromatin accessibility mapping method has been ATAC-seq (Buenrostro et al., 2013), in which the Transposon 5 (Tn5) cut-and-paste transposition reaction inserts sequencing adapters in the most accessible genomic regions (tagmentation). Because tagmentation creates sequencing libraries simultaneous with insertion into accessible sites, ATAC-seq is simple and fast, and successively improved ATAC-seq protocols have enhanced its popularity (Corces et al., 2016 and 2017).


Despite the utility of chromatin accessibility mapping, the mechanistic basis for chromatin accessibility itself has remained incompletely understood. In contrast to the simplistic designation of chromatin as being “open” or “closed,” recent work has shown that the median difference between an accessible and a non-accessible site in DNA is estimated to be only ~20%, with no sites completely accessible or inaccessible in a population of cells (Chereji et al., 2019; Oberbeckmann et al., 2019). To better understand this nuanced interpretation of chromatin accessibility, we have recently applied our Cleavage Under Targets & Tagmentation (CUT&Tag) method for antibody-tethered in situ tagmentation of chromatin to explore the mechanistic basis for chromatin accessibility (Henikoff et al., 2020). CUT&Tag uses a fusion protein between Protein A, which binds to the chromatin-bound antibody, and Tn5, which binds to adjacent DNA, and tagmentation occurs upon activation with Mg++ (Kaya-Okur et al., 2019). To suppress artifactual tagmentation of untargeted accessible chromatin, we performed all steps from pA-Tn5 fusion protein binding through tagmentation in the presence of 300 mM NaCl, which reduces non-specific DNA binding of the transposase. In the course of optimizing a simplified single-tube protocol, CUT&Tag-direct (Kaya-Okur et al., 2020), we serendipitously observed that simply reducing the ionic concentration during antibody-targeted tagmentation greatly increased the tendency of tethered Tn5 to tagment accessible chromatin near particular histone modifications (Henikoff et al., 2020). Preferential tagmentation of accessible chromatin only occurred when using antibodies against H3K4me2 and H3K4me3 and not for other histone modifications or variants. Because H3K4me2 flanks both promoters and enhancers genome-wide, the attraction of antibody-tethered Tn5 to nearby accessible DNA regions shifted the preferred sites of tagmentation from the nucleosomes bordering the Nucleosome-Depleted Region (NDR) to the NDR itself. Remarkably, practically all transcription-coupled accessible sites corresponded to ATAC-seq sites and vice-versa, upstream of paused RNA Polymerase II (RNAPII). Because of the close correspondence between the resulting “CUTAC” (Cleavage Under Targeted Accessible Chromatin) maps and DNaseI and ATAC-seq chromatin accessibility maps, we concluded that chromatin accessibility is driven by RNAPII transcriptional initiation (Henikoff et al., 2020), supporting previous suggestions that active promoters and enhancers are characterized by the same regulatory architecture (Andersson et al., 2015; Arnold et al., 2019).


In our original CUTAC study, we described three different modifications of the CUT&Tag-direct protocol for accessible site mapping: tagmentation in MgCl2 with a 20-fold dilution of 300 mM NaCl and pA-Tn5 (or commercial pAG-Tn5 with both Protein A and Protein G IgG specificities), removal of excess pAG-Tn5 before low-salt tagmentation, and low-salt tagmentation following the 300 mM wash step. We have adopted post-wash tagmentation, which follows the same steps as in the original CUT&Tag-direct protocol (Kaya-Okur et al., 2020), changing only the tagmentation buffer composition. As reported here, the application of this CUTAC protocol to the initiation form of RNAPII results in precise chromatin accessibility maps with exceptionally high signal-to-noise. The improvement obtained by tethering to the transcriptional machinery itself further supports the transcription-coupled basis for chromatin accessibility at enhancers and promoters.


CUT&Tag and CUTAC can be performed simultaneously in a single day from previously frozen native or lightly cross-linked nuclei through to purified sequencing-ready libraries, with all steps carried out in single PCR tubes. We present a simplified protocol where all steps from nuclei to purified sequencing-ready libraries are performed on a home benchtop using surplus equipment and non-toxic reagents (Figure 1). Our CUTAC results using an antibody to the Serine-5-phosphorylated initiation form of the repeated heptameric C-terminal domain of the largest RNAPII subunit (RNAPIIS5P) compare favorably with the best ATAC-seq data while providing a genome-wide map of the initiation form of RNAPII. The simplicity and affordability of the protocol make it equally suitable for a laboratory, home, or classroom environment.


Materials and Reagents

  1. Disposable tips (e.g., Rainin 1 ml, 200 µl, 20 µl)

  2. Disposable centrifuge tubes for reagents (15 ml or 50 ml)

  3. Standard 1.5 ml microfuge tubes

  4. 0.5 ml maximum recovery PCR tubes (e.g., Fisher, catalog number: 14-222-294)

  5. Phosphate-buffered saline (Fisher cat. no. BP3994)

  6. 16% (w/v) formaldehyde (10 × 1 ml ampules, Thermo Fisher, catalog number: 28906)

  7. 1.25 M glycine (Sigma-Aldrich, catalog number: G7126)

  8. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, catalog number: D4540)

  9. Cell culture (e.g., human K562 cells)

  10. Concanavalin A (ConA)-coated magnetic beads (Bangs Laboratories, catalog number: BP531)

  11. Distilled, deionized, or RNAse-free H2O (dH2O; e.g., Promega, catalog number: P1197)

  12. 1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.9 (HEPES (K+); Sigma-Aldrich, catalog number: H3375)

  13. 1 M Manganese Chloride (MnCl2; Sigma-Aldrich, catalog number: 203734)

  14. 1 M Calcium Chloride (CaCl2; Fisher, catalog number: BP510)

  15. 1 M Potassium Chloride (KCl; Sigma-Aldrich, catalog number: P3911)

  16. Roche Complete Protease Inhibitor EDTA-Free tablets (Sigma-Aldrich, catalog number: 5056489001)

  17. 1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.5 (HEPES (Na+); Sigma-Aldrich, catalog number: H3375)

  18. 5 M Sodium chloride (NaCl; Sigma-Aldrich, catalog number: S5150-1L)

  19. 2 M Spermidine (Sigma-Aldrich, catalog number: S0266)

  20. 0.5 M Ethylenediaminetetraacetic acid (EDTA; Research Organics, catalog number: 3002E)

  21. 200x Bovine Serum Albumen (BSA; NEB, catalog number: B9001S)

  22. Antibody to an epitope of interest for CUT&Tag

    Because in situ binding conditions are more like those for immunofluorescence (IF) than those for ChIP, we suggest choosing IF-tested antibodies if CUT&RUN/Tag-tested antibodies are not available.

  23. CUTAC control antibody to RNA Polymerase II Phospho-Rpb1 CTD Serine-5 phosphate (RNAPIIS5P) or histone H3K4me2. We have obtained excellent results with these rabbit monoclonal antibodies:

    Phospho-Rpb1 CTD (Ser5) (Cell Signalling Technology, catalog number: 13523 (D9N5I))

    H3K4me2 (Epicypher, catalog number: 13-0027)

  24. Secondary antibody, e.g., guinea pig α-rabbit antibody (Antibodies-Online, catalog number: ABIN101961) or rabbit α-mouse antibody (Abcam, catalog number: ab46540)

  25. Protein A/G-Tn5 (pAG-Tn5) fusion protein loaded with double-stranded adapters with 19mer Tn5 mosaic ends (Epicypher, catalog number: 15-1117)

  26. 1 M Magnesium Chloride (MgCl2; Sigma-Aldrich, catalog number: M8266-100G)

  27. 1 M [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS) pH 8.5 (with NaOH)

  28. 1,6-hexanediol (Sigma-Aldrich, catalog number: 240117-50G)

  29. N,N-dimethylformamide (Sigma-Aldrich, catalog number: D-8654-250 ml)

  30. NEBNext 2× PCR Master mix (ME541L)

  31. PCR primers: 10 µM stock solutions of i5 and i7 primers with unique barcodes [Buenrostro, J.D. et al., Nature 523:486 (2015)] in 10 mM Tris pH 8. Standard salt-free primers may be used. We do not recommend Nextera or NEBNext primers.

  32. 10% Sodium dodecyl sulfate (SDS; Sigma-Aldrich, catalog number: L4509)

  33. 10% Triton X-100 (Sigma-Aldrich, catalog number: X100)

  34. SPRI paramagnetic beads (e.g., HighPrep PCR Cleanup Magbio Genomics, catalog number: AC-60500)

  35. 10 mM Tris-HCl pH 8.0

  36. Ethanol (Decon Labs, catalog number: 2716)

  37. Nuclei Extraction 1 (NE1) buffer (see Recipes)

  38. Wash buffer (see Recipes)

  39. Binding buffer (see Recipes)

  40. Antibody buffer (see Recipes)

  41. 300-wash buffer (see Recipes)

  42. CUTAC Tagmentation buffer (see Recipes)

  43. TAPS wash buffer (see Recipes)

  44. 0.1% SDS Release solution (see Recipes)

  45. 0.67% Triton neutralization solution (see Recipes)

Equipment

  1. -80°C freezer

  2. Chilling device (e.g., metal heat blocks on ice or cold packs in an ice cooler)

  3. Pipettors (e.g., Rainin Classic Pipette 1 ml, 200 µl, 20 µl, and 10 µl)

  4. Strong magnet stand (e.g., Miltenyi Macsimag separator, catalog number: 130-092-168)

  5. Vortex mixer (e.g., VWR Vortex Genie)

  6. Minicentrifuge (e.g., VWR Model V)

  7. Tube rotator (e.g., Barnstead/Thermolyne 400110)

  8. PCR thermocycler (e.g., Bio-Rad/MJ PTC-200)



    Figure 1. A home workbench for CUT&Tag. Photo of the home workbench setup used for all experiments presented using this protocol. A typical experiment begins by mixing cells with activated ConA beads in up to 32 single PCR tubes, with all liquid changes performed on the magnet stand. The only tube transfer is the removal of the purified sequencing-ready libraries from the SPRI beads to fresh tubes for Tapestation analysis and DNA sequencing. The total time from thawing frozen nuclei until elution from SPRI beads is ~8 h.

Software

  1. Bowtie2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml

  2. Calibration script https://github.com/Henikoff/Cut-and-Run/blob/master/spike_in_calibration.csh

Procedure



Figure 2. Scheme for simultaneous CUT&Tag and (H3K4me2 or RNAPIIS5P) CUTAC. CUT&Tag-direct is performed in situ in single PCR tubes with Concanavalin A (ConA) bead-bound nuclei that remain intact throughout the protocol during successive liquid changes, incubations and washes, 12 cycles of PCR amplification, and one SPRI bead cleanup. CUTAC is performed identically except that low-salt conditions are used for tagmentation. H3K4me2 CUTAC maps accessible sites near H3K4me2/3-marked (starred) nucleosome tails, which are methylated by the conserved Set1 lysine methyltransferase. The complex that includes Set1 associates with the initiation form of RNAPII, which is heavily phosphorylated on Serine-5 of the heptameric C-terminal domain repeat units on the largest RNAPII subunit (RNAPIIS5P). For RNAPIIS5P CUTAC, pA-Tn5 is anchored directly to RNAPIIS5 phosphates (starred). Whereas CUT&Tag is suitable for any chromatin epitope, CUTAC is specific for H3K4me2, H3K4me3, and RNAPIIS5P. The only other difference between the protocols is that tagmentation is performed in the presence of 300 mM NaCl for CUT&Tag and in a low ionic strength buffer for CUTAC.


  1. Prepare and (optionally) lightly fix nuclei and cryopreserve (1 h in the lab)

    1. Harvest fresh culture(s) in a conical centrifuge tube (15 ml or 50 ml) at room temperature and count cells.

    2. Centrifuge for 3 min at 600 × g in a swinging bucket rotor at room temperature and drain the liquid.

    3. Resuspend in 1 volume of PBS (relative to starting culture) at room temperature by pipetting up and down.

    4. Centrifuge for 3 min at 600 × g in a swinging bucket rotor at room temperature and drain the liquid.

    5. Resuspend in 1/2 volume (relative to starting culture) of ice-cold NE1 buffer with gentle vortexing. Let sit on ice for 10 min.

    6. Centrifuge for 4 min at 1,300 × g at 4°C in a swinging bucket rotor and drain liquid by pouring off and inverting onto a paper towel for a few seconds.

    7. Resuspend in 1/2 volume of PBS. For unfixed nuclei, skip to Step A11.

    8. While gently vortexing, add 16% formaldehyde to 0.1% (e.g., 62 μl to 10 ml) and incubate at room temperature for 2 min.

      Note: Light fixation reduces the tendency of cells or nuclei to clump in the 300-wash buffer but can interfere with the binding of some antibodies, reducing yield.

    9. Stop cross-linking by adding 1.25 M glycine to twice the molar concentration of formaldehyde (e.g., 600 μl to 10 ml).

    10. Centrifuge for 4 min at 1,300 × g at 4°C and drain the liquid by pouring off and inverting onto a paper towel for a few seconds.

    11. Resuspend in Wash buffer to a concentration of ~1 million cells per ml. Check nuclei using ViCell or a cell counter slide.

    12. Nuclei may be slowly frozen by aliquoting 900 μl into cryogenic vials containing 100 μl of DMSO, mixed well, then placed in a Mr. Frosty container filled to the line with isopropanol and placed in a -80°C freezer overnight and stored at -80°C long term.

      Note: We have found that good results are obtained using native or cross-linked cells even after being stored in the freezer compartment of a side-by-side refrigerator for >6 months.


  2. Prepare Concanavalin A-coated beads (15 min)

    1. Resuspend and withdraw enough of the ConA bead slurry, ensuring that there will be 3.5 μl for each final sample of up to ~50,000 mammalian cells, which yield ≥50% K562 nuclei using this protocol. Transfer the ConA bead slurry into 1 ml of Binding buffer in a 1.5 ml tube.

      Note: This protocol has been used for up to 16 samples (60 µl beads) in 1 ml or 32 samples (120 µl beads) in 2 ml Binding buffer (in a 2 ml tube).

    2. Mix by pipetting. Place the tube on a magnet stand to clear (~1 min).

    3. Withdraw the supernatant completely and remove the tube from the magnet stand. Add 1 ml Binding buffer and mix by pipetting up and down.

    4. Place on the magnet stand to clear, remove and discard the supernatant, and resuspend in 60 µl Binding buffer (3.5 μl per sample).


  3. Bind nuclei to ConA beads (15 min)

    1. Thaw a frozen aliquot of nuclei at room temperature, for example, by placing in a 20 ml beaker of water.

      Note: The CUTAC control can use either native or lightly cross-linked nuclei, preferably prepared as previously described (Kaya-Okur et al., 2020). Do not use whole cells, which require a detergent and may also inhibit the PCR.

    2. Transfer the thawed nuclei suspension in aliquots of no more than ~50,000 starting mammalian cells to each thin-wall 0.5 ml PCR tube and mix with 3.5 µl ConA beads. Attach to the Tube rotator and rotate at room temperature for 10 min.

      Note: Nuclei prepared according to the recommended protocol (Kaya-Okur et al., 2020) have been resuspended in Wash buffer. Beads can be added directly to the aliquot for binding and then transferred to PCR tubes, ensuring that no more than 5 µl of the original ConA bead suspension is present in each PCR tube for single-tube CUT&Tag. Using more than ~50,000 mammalian nuclei or >5 µl Con A beads per sample may inhibit the PCR.

    3. Place the tubes on the magnet stand to clear and remove and discard the supernatant.

      Note: In low-retention PCR tubes, surface tension will cause bead-bound cells to slide down to the bottom of the tube at this step. To avoid beads being aspirated with the supernatant, set the pipette to a volume that is 5 µl less than the total volume to be removed. Use a careful second draw with a 20 µl pipette tip and remove as much supernatant as possible without aspirating beads.


  4. Bind primary antibody (1 h)

    1. For each CUT&Tag and CUTAC sample, mix the primary antibody 1:50-1:100 with Antibody buffer. Resuspend beads in 25 µl per sample with gentle vortexing.

      Note: We use 1:50-1:100 antibody dilutions by default or the manufacturer’s recommended concentration for immunofluorescence. CUTAC works best using either an RNA Polymerase II CTD-phosphorylated antibody (Ser5P > Ser2P/Ser5P > Ser2P) or an α-H3K4me2 antibody. α-H3K4me3 also works but is less efficient and is depleted at enhancer sites. Several antibodies to other histone epitopes have been tested, including α-H3K4me1, α-H3K36me3, α-H3K27ac, and α-H2A.Z, but all have failed.

    2. Place on a rotator at room temperature and incubate 1-2 h.

      Notes:

      1. Volumes up to 50 µl will remain in the tube bottom by surface tension during rotation, avoiding the need for a quick spin before the next step.

      2. After incubation, the tubes can be stored overnight at 4°C.

      3. An optional negative control is performed by omitting the primary antibody.


  5. Bind secondary antibody (1 h)

    1. Place tubes on the magnet stand to clear and remove and discard the supernatant.

      Note: Protein in the antibody solution improves bead adherence to the tube wall, allowing for complete removal of the liquid without dislodging the beads by doing two successive draws with a 20 µl pipettor set for maximum volume while being careful not to dislodge the beads by surface tension during the second draw.

    2. Mix the secondary antibody 1:100 in Wash buffer and add 25 µl per sample while gently vortexing to allow the solution to dislodge the beads from the sides.

      Notes:

      1. Calculate how much volume of diluted Antibody is necessary by multiplying the number of samples by 30 µl (which is 25 µl per sample plus overage for pipetting).

      2. The secondary antibody step is required for CUT&Tag to increase the number of Protein A binding sites for each bound antibody. We have found that without the secondary antibody, the efficiency is very low.

    3. Place the tubes on a rotator and rotate at room temperature for 0.5-1 h.

    4. After a quick spin (< 500 × g or just enough to remove the liquid from the sides of the tube), place the tubes on the magnet stand to clear and remove and discard the supernatant with two successive draws, using a 20 µl tip with the pipettor set for maximum volume.

    5. With the tubes still on the magnet stand, carefully add 500 µl of Wash buffer. The surface tension will cause the beads to slide up along the side of the tube closest to the magnet.

    6. Slowly remove 470 µl of supernatant with a 1 ml pipette tip without disturbing the beads.

      Note: To remove the supernatant, set the pipettor to 470 µl, and keep the plunger depressed while lowering the tip to the bottom. The liquid level will rise to near the top, completing the wash. Then ease off on the plunger until the liquid is withdrawn and remove the pipettor. During liquid removal, the surface tension will drag the beads down the tube. A small drop of liquid that is left behind will be removed in the next step.

    7. After a quick spin (<500 × g or just enough to remove the liquid from the sides of the tube), place the tubes back into the magnet stand and remove the remaining supernatant with a 20 µl pipettor, multiple times if necessary, to remove the entire supernatant without disturbing the beads. Proceed immediately to the next step.


  6. Bind pA-Tn5 adapter complex (1.5 h)

    1. Mix pAG-Tn5 pre-loaded adapter complex in 300-wash buffer following the manufacturer's instructions.

    2. Pipette in 25 µl per sample of the pA-Tn5 mix while vortexing and invert by rotation to ensure that beads adhering to the sides near the top of the top are resuspended.

      Note: When using the recommended Macsimag magnet stand, dislodging the beads after resuspending in pA-Tn5 can be done by removing the plexiglass tube holder from the magnet and, with fingers on top to prevent the tubes from opening or falling out, inverting by rotating sharply a few times.

    3. After a quick spin (<500 × g), place the tubes on a rotator at room temperature for 1-2 h.

    4. After incubating in the rotator, perform a quick spin and place the tubes in the magnet stand.

    5. Carefully remove the supernatant using a 20 µl pipettor twice to avoid disturbing the beads.

    6. With the tubes still on the magnet stand, add 500 µl of the 300-wash buffer.

    7. Slowly withdraw 470 µl with a 1 ml pipette tip without disturbing the beads as in Step D6.

    8. After a quick spin, place the tubes back on the magnet stand and remove and discard the supernatant with a 20 µl pipettor using multiple draws. Proceed immediately to the next step.


  7. Tagmentation and particle release (2.5 h) (Figure 2)

    1. Tagmentation:

      1. CUT&Tag samples only: Resuspend the bead/nuclei pellet in 50 µl CUT&Tag Tagmentation buffer (10 mM mM MgCl2 in 300-wash buffer) while vortexing or inverting by rotation to allow the solution to dislodge most or all the beads as in Step E2.

      2. CUTAC samples only: Resuspend the bead/nuclei pellet in 50 µl of either CUTAC-tag or CUTAC-hex Tagmentation buffer while vortexing or inverting by rotation to allow the solution to dislodge most or all the beads as in Step D6.

        Note: 10% 1,6-hexanediol or N,N-dimethylformamide compete for hydrophobic interactions and result in improved tethered Tn5 accessibility and library yield at the expense of slightly increased background.

    2. After a quick spin (<500 × g), incubate at 37°C for 1 h (20 min for CUTAC) in a PCR cycler with a heated lid. Hold at 8°C.

    3. Place tubes on the magnet stand and remove and discard the supernatant with a 20 µl pipettor using multiple draws, then resuspend the beads in 50 µl TAPS wash buffer and invert by rotation as in Step D6.

    4. After a quick spin, place tubes on the magnet stand and remove and discard the supernatant with a 20 µl pipettor using multiple draws.

    5. Resuspend the beads in 5 µl 0.1% SDS Release solution using a fresh 20 µl pipette tip to dispense while wetting the sides of the tubes to recover the fraction of beads sticking to the sides.

      Note: Rolling the tube back and forth rapidly between thumb and forefinger while brushing the pipette tip along the sides of the tube will effectively wet the beads; follow by a quick spin to bring most of the beads to the bottom.

    6. After a quick spin (<500 × g), incubate at 58°C for 1 h in a PCR cycler with a heated lid to release pA-Tn5 from the tagmented DNA.


  8. PCR (1 h)

    1. To the PCR tube containing the bead slurry, add 15 µl of Triton neutralization solution + 2 µl of 10 µM Universal or barcoded i5 primer + 2 µl of 10 µM uniquely barcoded i7 primers, using a different barcode for each sample. Vortex on full speed and place tubes in the metal tube holder on ice.

      Note: Indexed primers are described by Buenrostro et al. (2015). We do not recommend Nextera or NEB primers which might not anneal efficiently using this PCR protocol.

    2. Add 25 µl NEBnext (non-hot start), vortex to mix, and perform a quick spin. Place the tubes immediately in the thermocycler and proceed immediately with the PCR.

    3. Begin the cycling program with a heated lid on the thermocycler:

      1. Cycle 1: 58°C for 5 min (gap filling)

      2. Cycle 2: 72°C for 5 min (gap filling)

      3. Cycle 3: 98°C for 30 s

      4. Cycle 4: 98°C for 10 s

      5. Cycle 5: 60°C for 10 s

      6. Repeat Cycles 4-5 11 times

      7. 72°C for 1 min and hold at 8°C

      Notes:

      1. To minimize the contribution of large DNA fragments and excess primers, the PCR should be performed for no more than 12-14 cycles, preferably with a 10 s 60-63°C combined annealing/extension step as described above in Step H3e.

      2. The cycle times are based on using a conventional Peltier cycler (e.g., Bio-Rad/MJ PTC200), in which the ramping times (3°C/s) are sufficient for annealing to occur as the sample cools from 98°C to 60°C. Therefore, the use of a rapid cycler with a higher ramping rate will require either reducing the ramping time or other adjustments to assure annealing.

      3. Do not add extra PCR cycles to see a signal by capillary gel electrophoresis (e.g., Tapestation). If there is no nucleosomal ladder for the H3K27me3 positive control, you may assume that CUT&Tag failed, but observing no signal for a sparse chromatin protein such as a transcription factor is normal, and the barcoded sample can be concentrated for mixing with the pool of barcoded samples for sequencing. Extra PCR cycles reduce the complexity of the library and may result in an unacceptably high level of PCR duplicates.

      4. Cycle 3 (98 °C) may be extended from 30 sec to 5 min for cross-linked samples to ensure complete cross-link reversal.


  9. Post-PCR cleanup (30 min)

    1. After the PCR program ends, remove tubes from the thermocycler and add 65 μl of SPRI beads (ratio of 1.3 μl of SPRI beads to 1 μl of PCR product). Mix by pipetting up and down.

    2. Let sit at room temperature 5-10 min.

    3. Place on the magnetic stand for a few minutes to allow the solution to clear.

    4. Remove and discard the supernatant.

    5. Keeping the tubes in the magnetic stand, add 200 μl of 80% ethanol.

    6. Completely remove and discard the supernatant.

    7. Repeat Steps I5-I6.

    8. Perform a quick spin and remove the remaining supernatant with a 20 μl pipette, avoiding air drying the beads by proceeding immediately to the next step.

    9. Remove from the magnet stand, add 22 µl 10 mM Tris-HCl pH 8, and vortex at full speed. Let sit for 5 min to1 h.

    10. Place on the magnet stand and allow to clear.

    11. Remove the liquid to a new 1.5 ml tube with a pipette, avoiding transfer of beads.


  10. Tapestation analysis (Figure 3) and DNA sequencing

    1. Determine the size distribution and concentration of libraries by capillary electrophoresis using an Agilent 4200 TapeStation with D1000 reagents or equivalent.

      Note: We use the quantification by Tapestation to estimate library concentration and dilute each library to 2 nM before pooling based on fragment molarity in the 175-1,000 bp range. The concentration 2 nM has been determined empirically as the optimal library concentration used in the HiSeq by the Fred Hutch Genomics Shared Resource.

    2. Mix barcoded libraries to achieve equal representation as desired, aiming for a final concentration as recommended by the manufacturer. After mixing, perform an SPRI bead cleanup if needed to remove any residual PCR primers.

    3. Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions. For maximum economy, paired-end PE25 is more than sufficient for mapping to large genomes.

      Note: Using paired-end 25 × 25 sequencing on a HiSeq 2-lane rapid run flow cell, we obtain ~300 million total mapped reads, or ~3 million per sample when there are 96 samples mixed to obtain approximately equal molarity.



      Figure 3. Tapestation profiles for a low-cell-number RNAPIIS5P CUTAC experiment. Tagmentation was performed for 20 min at 37°C in CUTAC-hex buffer. Representative tracks for these samples are shown in Figure 4A.

Data analysis

  1. Align paired-end reads to hg19 using Bowtie2 version 2.3.4.3 with options: --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For mapping E. coli carry-over fragments, we also use the --no-overlap --no-dovetail options to avoid possible cross-mapping of the experimental genome to that of the carry-over E. coli DNA that is used for calibration. Tracks are made as bedgraph files of normalized counts, which are the fraction of total counts at each basepair scaled by the size of the hg19 genome.

    Note: To calibrate samples in a series for samples done in parallel using the same antibody, we use counts of E. coli fragments carried over with the pA-Tn5, as for an ordinary spike-in. Our sample script in Github can be used to calibrate based on either a spike-in or E. coli carry-over DNA.

  2. Our CUT&Tag Data Processing and Analysis Tutorial provides step-by-step guidance for mapping and analysis of CUT&Tag sequencing data. Most data analysis tools used for ChIP-seq data, such as bedtools, Picard, and deepTools, can be used on CUT&Tag data (Figures 3-5). Analysis tools designed specifically for CUT&RUN/Tag data include the SEACR peak caller, also available as a public web server, and CUT&RUNTools.



    Figure 4. Accessible DNA corresponds to binding sites of the initiating form of RNA Polymerase II (RNAPII). A. Tracks show profiles of the Chromosome 1 histone gene cluster, with 12 small intronless genes expressed at high levels in all dividing cells. Whereas RNAPIIS5P CUT&Tag shows broad enrichment over each of the genes, the CUTAC protocol applied to the RNAPIIS5P epitope, either native (RNAPIIS5P CUTAC-N) or cross-linked (RNAPIIS5P CUTAC-X), yields sharp promoter delineation, better than H3K4me2 CUTAC ± 1,6-hexanediol (turquoise) or the best K562 ATAC-seq datasets (green), all downsampled to 3.2 million mapped fragments. Note the 10-fold difference in scale between RNAPIIS5P CUTAC (0-1500 and K4me2-CUTAC/ATAC (0-150). Similar results were obtained for three mixed-lineage leukemia cell lines (KOPN8, SEM, and RS411) and H1 embryonic stem cells down to ~2,000 cells. No changes were made to the protocol for low cell numbers. Numbers in parentheses are estimated library sizes in millions of mapped paired-end reads. B-D. RNAPIIS5P occupies sites of accessible chromatin in K562 cells. B. Left to right: K4me2 CUT&Tag, K4me2 CUTAC, RNAPIIS5P CUT&Tag, RNAPIIS5P CUTAC, Omni-ATAC, and Fast-ATAC datasets were downsampled to 3.2 million fragments and aligned over ATAC-seq peaks called using MACS2 on data generated by the ENCODE project (ATACENCODE). C. Same as (A) except using only subnucleosome-sized fragments (≤120 bp). CUTAC RNAPIIS5P sites are virtually indistinguishable from high-quality ATAC-seq data, directly demonstrating that ATAC-seq maps sites of the initiation form of RNA Pol II. D. Same as (A) except using only >120 bp fragments. ENCODE ATAC-seq fragments were downsampled to 3.2 million, ChrM (mitochondrial DNA) was removed, and MACS2 was used to call peaks. Heatmaps are centered over ENCODE ATAC-seq peak summits and ordered by occupancy over the 5 kb span displayed. Fast-ATAC is an improved version of ATAC-seq that reduces mitochondrial reads (Corces et al., 2016), and Omni-ATAC is an improved version that additionally improves the signal-to-noise ratio (Corces et al., 2017). ATAC_ENCODE is the current ENCODE standard (Consortium et al., 2020).



    Figure 5. RNAPIIS5P CUTAC shows high sensitivity and specificity. Mapped fragments from the indicated K562 datasets were sampled, and peaks were called using MACS2. A) Number of peaks (left) and B) fraction of reads in peaks for CUT&Tag (triangles) and CUTAC (squares) profiles for H3K4me2, RNAPIIS5 (initiation form), RNAPIIS2P (elongation form), RNAPIIS2P/S5P, and Omni-ATAC (green). CUTAC for RNAPIIS5P shows the best sensitivity (most peaks at low sampling) and the best signal-to-noise (highest FRiP at all sampling levels). Tagmentation was for 10 min at 37°C in CUTAC-tag buffer.

Recipes

  1. Nuclei Extraction 1 (NE1) buffer

    Mix 1 ml of 1M HEPES-KOH pH 7.9, 500 μl of 1 M KCl, 12.5 μl of 2 M spermidine, 500 μl of 10% Triton X-100, and 10 ml of glycerol in 38 ml dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free.

  2. Wash buffer

    Mix 1 ml of 1 M HEPES pH 7.5, 1.5 ml of 5 M NaCl, and 12.5 μl of 2 M spermidine, bring the final volume to 50 ml with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4°C for up to 2 days.

  3. Binding buffer

    Mix 200 μl of 1M HEPES-KOH pH 7.9, 100 μl of 1 M KCl, 10 μl of 1M CaCl2, and 10 μl 1 M MnCl2, and bring the final volume to 10 ml with dH2O. Store the buffer at 4°C for up to several months.

  4. Antibody buffer

    Mix 5 µl of 200× BSA with 1 ml Wash buffer and chill on ice. BSA is present in some but not all antibody solutions, and 0.1% BSA in this buffer helps prevent bead loss during later steps.

  5. 300-wash buffer

    Mix 1 ml of 1 M HEPES pH 7.5, 3 ml of 5 M NaCl, and 12.5 μl of 2 M spermidine, bring the final volume to 50 ml with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store at 4°C for up to 2 days.

  6. CUT&Tag Tagmentation buffer

    Mix 1 ml of 300-wash buffer and 10 µl of 1 M MgCl2 (to 10 mM).

  7. CUTAC Tagmentation buffer

    1. CUTAC-tag: Mix 197 µl of dH2O, 2 µl of 1 M TAPS pH 8.5, and 1 µl of 1 M MgCl2 (10 mM TAPS and 5 mM MgCl2). Store the buffer at 4°C for up to 1 day.

    2. CUTAC-hex: Mix 97 µl of dH2O, 100 µl of 20% (w/v) 1,6-hexanediol, 2 µl of 1 M TAPS pH 8.5, and 1 µl of 1 M MgCl2 (10 mM TAPS, 5 mM MgCl2 10% 1,6-hexanediol). Store the buffer at 4°C for up to 1 day.

  8. TAPS wash buffer

    Mix 1 ml of dH2O, 10 µl of 1 M TAPS pH 8.5, 0.4 µl of 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA)

  9. 0.1% SDS Release solution

    Mix 10 µl of 10% SDS and 10 µl of 1 M TAPS pH 8.5 in 1 ml of dH2O

  10. 0.67% Triton neutralization solution

    Mix 67 µl of 10% Triton-X100 + 933 µl dH2O

Acknowledgments

This protocol was adapted from a previous publication (Henikoff et al., 2020) and expanded to include RNAPIIS5P CUTAC, with datasets available from GEO (GSE158327). We thank Terri Bryson and Christine Codomo for sample processing, the Fred Hutch Genomics Shared Resource for DNA sequencing, members of our laboratory for helpful discussions, and Paul Talbert for critically reading the manuscript. S. H. is an Investigator of the Howard Hughes Medical Institute. This work was supported by the Howard Hughes Medical Institute (S.H.), grants R01 HG010492 (S.H.) and R01 GM108699 (K.A.) from the National Institutes of Health, and an HCA Seed Network grant from the Chan-Zuckerberg Initiative (S.H.).

Competing interests

SH has filed patent applications related to this work. JGH and KA declare no competing interests.

References

  1. Andersson, R., Sandelin, A. and Danko, C. G. (2015). A unified architecture of transcriptional regulatory elements. Trends Genet 31(8): 426-433.
  2. Arnold, P. R., Wells, A. D. and Li, X. C. (2019). Diversity and Emerging Roles of Enhancer RNA in Regulation of Gene Expression and Cell Fate. Front Cell Dev Biol 7: 377.
  3. Auerbach, R. K., Euskirchen, G., Rozowsky, J., Lamarre-Vincent, N., Moqtaderi, Z., Lefrancois, P., Struhl, K., Gerstein, M. and Snyder, M. (2009). Mapping accessible chromatin regions using Sono-Seq. Proc Natl Acad Sci U S A 106(35): 14926-14931.
  4. Bownes, M. (1990). Preferential insertion of P elements into genes expressed in the germ-line of Drosophila melanogaster.Mol Gen Genet 222(2-3): 457-460.
  5. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. and Greenleaf, W. J. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10(12): 1213-1218.
  6. Buenrostro, J. D., Wu, B., Litzenburger, U. M., Ruff, D., Gonzales, M. L., Snyder, M. P., Chang, H. Y. and Greenleaf, W. J. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523(7561): 486-490.
  7. Chereji, R. V., Eriksson, P. R., Ocampo, J., Prajapati, H. K. and Clark, D. J. (2019). Accessibility of promoter DNA is not the primary determinant of chromatin-mediated gene regulation. Genome Res 29(12): 1985-1995.
  8. Corces, M. R., Buenrostro, J. D., Wu, B., Greenside, P. G., Chan, S. M., Koenig, J. L., Snyder, M. P., Pritchard, J. K., Kundaje, A., Greenleaf, W. J., Majeti, R. and Chang, H. Y. (2016). Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat Genet 48(10): 1193-1203.
  9. Corces, M. R., Trevino, A. E., Hamilton, E. G., Greenside, P. G., Sinnott-Armstrong, N. A., Vesuna, S., Satpathy, A. T., Rubin, A. J., Montine, K. S., Wu, B., Kathiria, A., Cho, S. W., Mumbach, M. R., Carter, A. C., Kasowski, M., Orloff, L. A., Risca, V. I., Kundaje, A., Khavari, P. A., Montine, T. J., Greenleaf, W. J. and Chang, H. Y. (2017). An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.Nat Methods 14(10): 959-962.
  10. Crawford, G. E., Holt, I. E., Mullikin, J. C., Tai, D., Blakesley, R., Bouffard, G., Young, A., Masiello, C., Green, E. D., Wolfsberg, T. G., Collins, F. S. and National Institutes Of Health Intramural Sequencing, C. (2004). Identifying gene regulatory elements by genome-wide recovery of DNase hypersensitive sites.Proc Natl Acad Sci U S A 101(4): 992-997.
  11. Dorschner, M. O., Hawrylycz, M., Humbert, R., Wallace, J. C., Shafer, A., Kawamoto, J., Mack, J., Hall, R., Goldy, J., Sabo, P. J., Kohli, A., Li, Q., McArthur, M. and Stamatoyannopoulos, J. A. (2004). High-throughput localization of functional elements by quantitative chromatin profiling. Nat Methods 1(3): 219-225.
  12. Giresi, P. G., Kim, J., McDaniell, R. M., Iyer, V. R. and Lieb, J. D. (2007). FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. Genome Res 17(6): 877-885.
  13. Gottschling, D. E. (1992). Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo.Proc Natl Acad Sci U S A 89(9): 4062-4065.
  14. Henikoff, S., Henikoff, J. G., Kaya-Okur, H. S. and Ahmad, K. (2020). Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation.Elife 9: e63274.
  15. Jack, R. S. and Eggert, H. (1990). Restriction enzymes have limited access to DNA sequences in drosophila chromosomes. EMBO J 9(8): 2603-2609.
  16. Kaya-Okur, H. S., Janssens, D. H., Henikoff, J. G., Ahmad, K. and Henikoff, S. (2020). Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15(10): 3264-3283.
  17. Kaya-Okur, H. S., Wu, S. J., Codomo, C. A., Pledger, E. S., Bryson, T. D., Henikoff, J. G., Ahmad, K. and Henikoff, S. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10(1): 1930.
  18. Consortium, E. P., Moore, J. E., Purcaro, M. J., Pratt, H. E., Epstein, C. B., Shoresh, N., Adrian, J., Kawli, T., Davis, C. A., Dobin, A., Kaul, R., Halow, J., Van Nostrand, E. L., Freese, P., Gorkin, D. U. et al. (2020). Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583(7818): 699-710.
  19. Oberbeckmann, E., Wolff, M., Krietenstein, N., Heron, M., Ellins, J. L., Schmid, A., Krebs, S., Blum, H., Gerland, U. and Korber, P. (2019). Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome.Genome Res 29(12): 1996-2009.
  20. Reeves, R. (1978). Nucleosome structure of Xenopus oocyte amplified ribosomal genes. Biochemistry 17(23): 4908-4916.
  21. Weintraub, H. and Groudine, M. (1976). Chromosomal subunits in active genes have an altered conformation. Science 193(4256): 848-856.

简介

[摘要]我们先前介绍了在目标与标签下切割(CUT&Tag ),这是一种表观基因组学分析方法,其中Tn5转座酶与目标染色质抗原决定簇的抗体结合作用可映射小样品和单细胞中的特定染色质特征。使用CUT&Tag可使完整的细胞或细胞核通透,然后依次添加一抗,二抗和与该抗体结合的嵌合蛋白A-转座酶融合蛋白。Mg ++的加入激活了转座酶,并将测序衔接子原位插入相邻的DNA中。此后,我们对CUT&Tag进行了改编也通过简单地使用组蛋白H3K4me2,H3K4me3的当修改所述转活化条件映射染色质访问性,或丝氨酸-5磷酸化的RNA聚合酶II的抗体。使用这些抗体,我们将可访问的DNA位点的标签重定向,以产生具有极高信噪比和分辨率的染色质可访问性图。所有的原子核扩增测序就绪库步骤在单个PCR管进行使用无毒试剂和廉价的设备,使我们简化战略,同时染色体分析和适合实验室无障碍映射,家庭工作台,或教室。


[背景] DNA中的辅助染色质景观的映射是首先描述45年前与观察DNA酶I超敏反应在转录活性的基因座(温特劳布和Groudine,1976) 。由于DNaseI优先切割缺少核小体的基因组区域,并且调控元件与非组蛋白染色质蛋白而不是核小体结合,因此DNaseI超敏位点定位已被用于表征遗传调控态势。染色质可接近性其他酶探针包括微球菌核酸酶(MNase )(Reeves的等人,1978) ,限制性内切酶(Jack和Eggert的,1990) ,转座(Bownes,1990) ,和DNA甲基转移酶(Gottschling等人,1992) 。随着全基因组读出平台的引入,超敏性位点定位变得更加常规化,首先是从微阵列开始,然后是短读DNA测序(Crawford等,2004; Dorschner等,2004)。染色质的可及性还利用交联染色质的物理破碎和差异回收作图,FAIRE (Giresi等,2007)和Sono-Seq (Auerbach等,2009)的基础。近年来,最流行的染色质可及性作图方法是ATAC-seq (Buenrostro等人,2013),其中转座子5(Tn5)剪切和粘贴转座反应在最易接近的基因组区域插入测序适配器(标签化)。由于标签化在插入可访问位点的同时创建了测序文库,因此ATAC-seq既简单又快速,并且相继改进的ATAC-seq协议增强了其受欢迎度(Corces等人,2016和2017)。

尽管染色质可访问性映射实用,但对染色质可访问性本身的机制基础仍不完全了解。与将染色质简单地命名为“开放”或“封闭”相反,最近的研究表明,DNA中可及和不可及位点之间的中值差异估计仅为〜20%,而没有位点完全访问或在细胞群不可访问(Chereji等人,2019; Oberbeckmann 。等人,2019) 。为了更好地理解染色质辅助功能的这种细致入微的诠释,我们最近应用了我们ç leavage ü的nDer牛逼具体目标和标签的心理状态(CUT及标签)的方法进行抗体系留在原位tagmentation染色,探讨染色质的无障碍机制基础的(参见Henikoff等。(2020年)。CUT&Tag在与染色质结合抗体结合的Protein A和与相邻DNA结合的Tn5之间使用了融合蛋白,当用Mg ++激活时就会发生标签化(Kaya-Okur等人,2019)。为了抑制未靶向可及染色质的人为标记,我们在300 mM NaCl存在下执行了从pA-Tn5融合蛋白结合到标记的所有步骤,这减少了转座酶的非特异性DNA结合。在优化的简化单管协议,的过程中CUT&标签直接(卡亚-奥库等人,2020) ,我们偶然观察到,简单地减少抗体靶向过程中的离子浓度tagmentation大大增加拴系的Tn5到的倾向tagment访问染色质接近特定的组蛋白修饰(Henikoff et al。,2020)。优惠tagmentation可接近的染色质的仅使用针对H3K4me2与H3K4me3的抗体时出现,并没有对其他组蛋白修饰或变体。由于H3K4me2在全基因组两侧都位于启动子和增强子的两侧,因此,与抗体相连的Tn5对附近可访问的DNA区域的吸引力将标记的首选位点从与核小体耗尽区(NDR)毗邻的核小体转移到NDR本身。值得注意的是,在暂停的RNA聚合酶II(RNAPII)的上游,几乎所有转录偶联的可及位点都对应于ATAC-seq位点,反之亦然。由于紧密对应的之间所产生的“CUTAC”(Ç leavage û的nDer Ť argeted甲ccessible Ç hromatin)映射和DNA酶I和ATAC- SEQ染色质可访问映射,我们的结论是,染色质访问性是由RNAPII转录起始驱动(参见Henikoff等人。,2020) ,支持以前的建议,活性的启动子和增强子由相同的调节特征架构(安德森等人,2015;阿诺德等人,2019) 。

在我们的最初的CUTAC研究中,我们所描述的三种不同的修改CUT&标签直接用于访问站点映射协议:tagmentation中的MgCl 2与一个300mM的NaCl和PA-Tn5转(或商业PAG-Tn5转的20倍稀释液与两个A蛋白和蛋白G IgG特异性),在低盐标记之前去除过量的pAG-Tn5以及在300 mM洗涤步骤后进行低盐标记。我们采用了洗后标记,其步骤与原始CUT&Tag-direct协议(Kaya-Okur等,2020)中的步骤相同,仅更改了标记缓冲液的成分。如在此报道,在此CUTAC协议RNAPII导致精确染色质可接近性起始形式的应用映射与异常高的信号-噪声。通过拴系到转录机制本身而获得的改进进一步支持了染色质在增强子和启动子处的可及性的转录偶联基础。

从先前冷冻的天然或轻度交联的细胞核到纯化的可测序的文库,CUT&Tag和CUTAC可以在一天内同时进行,所有步骤均在单个PCR管中进行。我们提供了一个简化的协议,其中从核到纯化的可测序文库的所有步骤均在家用台式机上使用多余的设备和无毒试剂进行(图1)。我们的CUTAC结果使用了最大RNAPII亚基(RNAPIIS5P)的重复七聚体C末端结构域的丝氨酸5磷酸化起始形式的抗体,与最佳ATAC-seq数据相比具有优势,同时提供了起始的全基因组图谱形式的RNAPII。该协议的简单性和经济性使它同样适合于实验室,家庭,或教室环境。

关键字:表观基因组分析, 染色质可及性, RNA聚合酶II, 组蛋白变化, 目标与标签



材料和试剂


一次性吸头(例如,的Rainin 1毫升,200微升,20微升)
用于试剂的一次性离心管(15毫升或50毫升)
标准1.5 ml微量离心管
0.5毫升最大回收PCR管(例如,费舍尔,目录号:14-222-294)
磷酸盐缓冲盐水(Fisher货号BP3994)
16%(w / v)甲醛(10 × 1毫升安瓿瓶,赛默飞世尔(Thermo Fisher),目录号:28906)
1.25 M甘氨酸(Sigma-Aldrich ,目录号:G7126)
二甲基亚砜(DMSO; Sigma-Aldrich ,目录号:D4540)
细胞培养物(例如,人的K562细胞)
包有伴刀豆球蛋白A(ConA )的磁珠(Bangs Laboratories ,目录号:BP531)
蒸馏水,去离子,或RNA酶-freeħ 2 O(卫生署2 ö ;例如,Promega公司,目录号:P1197)
1 M羟乙基哌嗪乙烷磺酸磺酸盐pH 7.9(HEPES(K + ); Sigma-Aldrich ,目录号:H3375)
1 M氯化锰(MnCl 2 ;西格玛奥德里奇(Sigma-Aldrich),目录号:203734)
1 M氯化钙(CaCl 2 ; Fisher ,目录号:BP510)
1 M氯化钾(KCl ; Sigma-Aldrich ,目录号:P3911)
罗氏Complete Protease Inhibitor EDTA-free片剂(Sigma-Aldrich ,目录号:5056489001)
1 M羟乙基哌嗪乙烷磺酸pH值7.5(HEPES(Na + ); Sigma-Aldrich ,目录号:H3375)
5 M氯化钠(NaCl; Sigma-Aldrich ,目录号:S5150-1L)
2 M亚精胺(Sigma-Aldrich ,目录号:S0266)
0.5 M乙二胺四乙酸(EDTA; Research Organics ,目录号:3002E)
200x牛血清蛋白(BSA ; NEB ,目录号:B9001S)
CUT&Tag感兴趣的抗原决定簇的抗体
由于现场结合的条件更喜欢那些免疫荧光(IF)比FO [R的ChIP ,我们建议您选择IF-测试的抗体,如果CUT&RUN /标签测试抗体不可用。


针对RNA聚合酶II Phospho-Rpb1 CTD丝氨酸5磷酸(RNAPIIS5P)或组蛋白H3K4me2的CUTAC对照抗体。这些兔单克隆抗体已获得出色的结果:
Phospho-Rpb1 CTD(Ser5)(细胞信号技术,目录号:13523(D9N5I))


H3K4me2 (Epicypher ,目录号13-0027 )


二抗,例如,豚鼠α-兔抗体(抗体-O nline ,目录号:ABIN101961)或兔α-小鼠抗体(Abcam ,目录号:ab46540)
载有带有19mer Tn5镶嵌末端的双链衔接子的蛋白A / G - Tn5(pAG-Tn5)融合蛋白(Epicypher ,目录号15-1117 )
1 M氯化镁(MgCl 2 ; Sigma-Aldrich,目录号:M8266-100G)
1 M [三(羟甲基)甲基氨基]丙烷磺酸(TAPS)pH 8.5(使用NaOH)
1,6-己二醇(Sigma-Aldrich ,目录号:240117-50G)
N,N-二甲基甲酰胺(Sigma-Aldrich ,目录号:D-8654-250 ml)
NEBNext 2 × PCR预混液(ME541L)
PCR引物:带有独特条形码的i5和i7引物的10 µM储备液[ Buenrostro ,JD等。,Nature 523:486(2015)]在10 mM Tris pH 8中使用。可以使用标准的无盐引物。我们不建议使用Nextera或NEBNext底漆。
10%十二烷基硫酸钠(SDS; Sigma-Aldrich,目录号:L4509)
10%Triton X-100(Sigma-Aldrich,目录号:X100)
SPRI顺磁珠(例如,HighPrep PCR纯化Magbio基因组学,目录号:AC-60500)
10毫米Tris-HCl pH 8.0
乙醇(Decon Labs,目录号:2716)
核提取1(NE1)缓冲液(请参阅食谱)
洗涤缓冲液(请参见食谱)
绑定缓冲区(请参阅食谱)
抗体缓冲液(请参阅食谱)
300洗涤缓冲液(请参阅配方)
CUTAC标签缓冲(请参阅食谱)
TAPS清洗缓冲液(请参见配方)
0.1%SDS释放解决方案(请参阅食谱)
0.67%Triton中和溶液(请参阅配方)


设备


-80°C冷冻室
冷却装置(例如,冰上的金属加热块或冰柜中的冰袋)
移液器(例如,的Rainin经典移液器1毫升,200微升,20微升,和10微升)
强力磁体支架(例如,Miltenyi Macsimag分离器,目录号:130-092-168)
涡流混合器(例如,VWR涡流精灵)
Minicentrifuge(例如,VWR模型V)
试管旋转器(例如,巴恩斯特德/ THERMOLYNE 400110)
PCR热循环仪(例如,Bio - Rad / MJ PTC-200)




图1 。CUT&Tag的家庭工作台。P给出使用该协议用于所有的实验家工作台设置的HOTO。一个典型的实验开始于将细胞与激活的ConA珠子混合在多达32个单个PCR管中,并在磁架上进行所有液体更换。唯一的试管转移是将纯化的可测序的文库从SPRI珠子移至新鲜的试管,以进行Tapestation分析和DNA测序。从融化冷冻核到从SPRI珠洗脱下来的总时间为〜8 h。


软件


Bowtie2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
校准脚本https://github.com/Henikoff/Cut-and-Run/blob/master/spike_in_calibration.csh


程序




图2.同时进行CUT&Tag和(H3K4me2或RNAPIIS5P)CUTAC的方案。CUT&标签直接进行原位与单个PCR管伴刀豆球蛋白A(刀豆)珠结合的核即在连续的液体的变化,温育和洗涤后,PCR扩增12个循环保持整个协议完整,和一个SPRI珠清理。除了将低盐条件用于标记外,CUTAC的执行方式相同。H3K4me2 CUTAC可绘制H3K4me2 / 3标记(加星标)核小体尾部附近的可及位点,这些尾部由保守的Set1赖氨酸甲基转移酶甲基化。包含Set1的复合物与RNAPII的起始形式相关联,RNAPII在最大RNAPII亚基(RNAPIIS5P)上七聚体C末端域重复单元的Serine-5上被严重磷酸化。对于RNAPIIS5P CUTAC,pA-Tn5直接锚固在RNAPIIS5磷酸盐(带星号)上。而CUT&标签适用于任何染色质表位,CUTAC是具体的H3K4me2,H3K4me3的,和RNAPIIS5P。的协议之间的唯一的其他不同的是,tagmentation在300的存在下进行毫氯化钠为CUT&标签和用于CUTAC低离子强度缓冲液中。


准备并(可选)轻轻固定细胞核和冷冻保存液(实验室中1小时)
在室温下,在锥形离心管(15 ml或50 ml)中收集新鲜培养物并计数细胞。
离心机为3分钟,在600 ×克在室温下吊桶式转头和漏的液体。
通过上下移液在室温下重悬于1体积的PBS中(相对于开始培养)。
离心机为3分钟,在600 ×克在室温下吊桶式转头和漏的液体。
重悬于1/2体积(相对于起始培养物)的冰冷缓冲液NE1温和涡旋。让我们坐冰为10分钟。
离心机为4分钟,在1 ,300 ×克在4℃下在吊桶式转头和漏极通过倾倒出并反转在纸巾上几秒钟的液体。
重悬于1/2体积的PBS中。对于未固定的原子核,请跳至步骤A 11。
同时轻轻涡旋,16%甲醛加入到0.1%(例如,62 μ升在室温下2分钟,10ml)中并孵育。
注意:光固定可以减少细胞或细胞核在300洗涤缓冲液中结块的趋势,但会干扰某些抗体的结合,从而降低产量。


停止通过阿迪交联纳克的1.25M甘氨酸甲醛(两次摩尔浓度例如,600 μ升〜10毫升)。
离心机为4分钟,在1 ,300 ×克在4℃和漏极的通过倒出和反相纸巾上几秒钟的液体。
重悬于洗涤缓冲液中至每毫升约100万个细胞的浓度。检查使用核ViCell或一个细胞计数滑块。
核可以由缓慢冷冻等分900 μ升到含有100低温小瓶μ升的DMSO,充分混合,然后放置在充满到用异丙醇行了雾先生容器并放置在-80℃冷冻器中过夜,并贮存于长期-80°C。
注意:我们发现,即使将天然细胞或交联细胞保存在并排冰箱的冷藏室中超过6个月,也能获得良好的效果。


准备伴刀豆球蛋白A包被的珠子(15分钟)
重悬和足够撤回的刀豆珠浆,确保将有3.5微升最多的至〜50000哺乳动物细胞每个最终样品,其产率≥50%K562细胞核使用该协议。Ť转让(BOT)的刀豆珠浆液到1ml的结合缓冲液在1.5ml管中。
注意:此方案已用于1 ml中的多达16个样品(60 µl珠子)或2 ml结合缓冲液(在2 ml管中)的32个样品(120 µl珠子)。


通过移液混合。将试管放在磁铁架上清理(〜1分钟)。
完全取出上清液,然后从磁力架上取下试管。加入1 ml结合缓冲液,上下移液混合。
在地方的磁体支架清除,取下并丢弃上清液,重悬于60微升结合缓冲液(3.5微升每个样品)。


将细胞核绑定到ConA磁珠(15分钟)
在室温下解冻冷冻的细胞核等分试样,例如,将其放入20 ml的烧杯中。
                                                        注意:CUTAC对照可以使用天然或轻度交联的核,最好按照先前的描述进行制备(Kaya-Okur等,2020)。不要使用完整的细胞,需要洗涤剂和可能也抑制PCR。


在不超过〜50000开始的哺乳动物细胞的等分试样转移解冻晶核悬浮液到每个薄壁0.5米升PCR管中并用3.5微升混合刀豆珠。附连到管ř otator并在室温下旋转10分钟。
注意:根据推荐规程(Kaya-Okur等,2020)制备的细胞核已重悬于洗涤缓冲液中。可以将珠子直接添加到等分试样中以进行结合,然后转移到PCR试管中,确保用于单管CUT&Tag的每个PCR试管中不超过5 µl的原始ConA珠子悬浮液。每个样品使用超过50,000个哺乳动物核或> 5 µl Con A珠子可能会抑制PCR。


将试管放在磁体支架上,以清除并去除并丢弃上清液。
注意:在低保留度PCR试管中,在此步骤中,表面张力会导致与小珠结合的细胞向下滑动至试管底部。为避免珠子被上清液吸走,将移液器的体积设置为比要去除的总体积小5 µl 。用20 µl移液器吸头小心地进行第二次抽吸,并在不吸出珠子的情况下尽可能多地去除上清液。


结合一抗(1小时)
对于每个CUT&Tag和CUTAC样品,将一抗1:50-1:100与抗体缓冲液混合。重悬珠子,每个样品25 µl,轻轻涡旋。
注意:默认情况下,我们使用1:50-1:100抗体稀释度s或制造商建议的免疫荧光浓度。使用RNA聚合酶II CTD磷酸化抗体(Ser5P> Ser2P / Ser5P> Ser2P)或α-H3K4me2抗体,CUTAC效果最佳。α-H3K4me3也可以工作,但效率较低,并且在增强子位点被耗尽。已测试了针对其他组蛋白表位的几种抗体,包括α-H3K4me1,α-H3K36me3 ,α-H3K27ac和α-H2A.Z ,但都失败了。


上的一个地方- [R在室温下孵育1-2小时otator。
注意小号:


在旋转过程中,由于表面张力,最大50 µl的体积将留在管底部,从而避免了在下一步之前快速旋转的需要。
孵育后,试管可在4°C下保存过夜。
通过省略一抗进行任选的阴性对照。


结合二抗(1小时)
将试管放在磁体支架上,以清除并去除并丢弃上清液。
注:蛋白质中的抗体溶液改善了胎圈粘附在管壁上,从而允许完全移除的所述液体而不逐出所述珠通过执行两个连续的平与20μl的移液管组为最大体积,同时小心不要移出由表面珠粒在第二次抽奖中的张力。


将二抗以1:100的比例在洗涤缓冲液中混合,并在每个样品中加入25 µl,同时轻轻涡旋以使溶液从侧面移走小珠。
注意小号:


计算稀释的抗体的多少体积乘以必要的由30个样本的数微升(这是25微升每样品加过量用于吸移)。
CUT&Tag需要第二抗体步骤,以增加每种结合抗体的蛋白质A结合位点的数量。我们发现,没有二抗,效率非常低。
放置在所述管- [R otator并在室温下旋转0.5-1小时。
快速旋转(<500 × g或刚好足以从管子的侧面除去液体)后,将管子放在磁体支架上,以连续两次抽吸(使用20 µl针尖)将其清除并除去并丢弃上清液。移液器设置为最大音量。
与管仍然在磁体支架,小心地加入500μl的洗涤缓冲液。表面张力将导致磁珠沿最靠近磁体的管子侧面向上滑动。
用1 ml移液器吸头缓慢除去470 µl上清液,而不会干扰珠子。
注意:要去除上清液,请将移液器设置为470 µl,并保持柱塞压下,同时将吸头降低到底部。液位将上升到顶部附近,完成清洗。然后放下柱塞,直到抽出液体,然后移开移液器。在除去液体的过程中,表面张力会将珠子向下拖动到管子上。下一步将除去一小滴残留的液体。


一个快速旋转后(<500 ×克或刚好足以除去从所述管的侧面上的液体),将管放回磁体支架,并用20除去剩余的上清液微升移液器,如果必要多次,以除去整个上清液不干扰珠子。立即进行下一步。


绑定pA-Tn5适配器复合体(1.5小时)
按照制造商的说明,将pAG-Tn5预装的适配器复合物在300洗涤缓冲液中混合。
用移液器吸取每个样品中25μl的pA-Tn5混合物,同时涡旋并旋转以确保附着在顶部顶部附近侧面的珠子重新悬浮。
注意:当使用推荐的Macsimag磁体支架,在PA-Tn5的再悬浮可以通过从磁体取出有机玻璃管夹持器来完成后移去所述珠和,在上面的手指,以防止从开口或掉出管,转化荷兰国际集团由急剧旋转几次。


一个快速旋转后(<500 ×克),将管上的[R在室温下otator 1个-2小时。
在孵化后[R otator,执行快速旋转并放置在试管磁铁立场。
用20 µl移液器两次小心除去上清液,以免干扰磁珠。
将试管仍放在磁力架上,加入500 µl 300洗涤缓冲液。
用1 ml移液器吸头缓慢取出470 µl,而不干扰步骤D6中的珠子。
快速旋转后,将试管放回磁体支架上,并用多次抽吸用20 µl移液器移出并丢弃上清液。立即进行下一步。


标记和颗粒释放(2.5小时)(图2)
标签:
CUT&标签样品只:重悬珠子/细胞核沉淀于50μl CUT&标签Ť破碎块缓冲器(10毫毫的MgCl 2中的300的洗涤缓冲液),同时涡旋或旋转反转以使该溶液以去除大部分或全部的珠子如步骤E2 。
仅CUTAC样品:将小珠/细胞核小球重悬于50 µl CUTAC标签或CUTAC-hex T混合缓冲液中,同时通过旋转涡旋或颠倒,使溶液如步骤D6所示移出大部分或所有小珠。
注:10%1 ,6 -hexanediol或N,对于疏水性相互作用N-二甲基甲竞争,并且导致改善拴系的Tn5可访问性和在稍微增加背景为代价库收率。


快速旋转(< 500 × g )后,在37孵育℃,用于与PCR循环仪1个小时(20分钟为CUTAC)一个加热盖。保持在8°C。
在地方管的磁体支架和取出并丢弃使用多个绘制一个20微升移液器上清,然后重悬在50μlTAPS珠粒洗涤缓冲液,并通过旋转倒置如步骤D6。
快速旋转后,将试管放在磁体支架上,并用多次抽吸用20 µl移液器除去并丢弃上清液。
使用新的20 µl移液器吸头将珠子重悬于5 µl 0.1%SDS释放溶液中,同时润湿试管的侧面,以回收附着在侧面的珠子部分。
注意:在拇指和食指之间快速来回滚动管子的同时,沿管子的侧面刷移液管尖端会有效地润湿珠子。然后快速旋转,将大部分珠子移至底部。


一个快速旋转后(< 500 ×克)中,i ncubate在58 ℃下用于与PCR循环仪1个小时一个加热盖以从释放PA-Tn5转tagmented DNA。


PCR(1小时)
向含有珠浆液,将该PCR管,添加15微升的的Triton和溶液+ 2微升10μM通用或条形码i5的引物+ 2的10个μM唯一条形码i7的引物微升,使用对于每个样品不同的条形码。以全速涡旋并将管子放入冰上的金属管支架中。
注意:索引引物由Buenrostro等描述。(2015年)。我们不建议使用该PCR方案可能无法有效退火的Nextera或NEB引物。


加入25 µl NEBnext (非热启动),涡旋混合,然后快速旋转。立即将试管放入热循环仪中,并立即进行PCR。
首先循环程序一个在热循环加热盖:
周期1:58°C持续5分钟(间隙填充)
周期2:72°C持续5分钟(间隙填充)
周期3:98°C持续30 s
周期4:98°C持续10 s
周期5:60°C持续10 s
重复循环4-5 11次
72°C 1分钟并保持在8°C
注意小号:


为了最大限度地减少大的DNA片段和过量的引物的贡献,所述PCR应该不超过12-14个循环来进行,优选用如上所述的10秒60-63℃,合并的退火/延伸步骤我Ñ小号TEP ħ 3e中。
循环时间是基于使用常规Peltier循环仪(例如,Bio - Rad / MJ PTC200)的,其中随着样品从98°C冷却到60°C的升温时间(3°C / s)足以进行退火℃。因此,使用具有较高斜率的快速循环仪将需要减少斜时间或进行其他调整以确保退火。
不添加额外的PCR循环以查看通过毛细管凝胶电泳(例如,信号,Tapestation )。如果没有H3K27me3阳性对照的核小体阶梯,则可以假设CUT&Tag失败,但是观察到稀疏染色质蛋白的信号(如转录因子)正常,则可以浓缩条形码化的样品以与条形码化的池混合用于测序的样品。额外的PCR循环降低了文库的复杂性,并可能导致PCR复制品的含量过高。
对于交联样品,可以将周期3(98°C)从30秒延长到5分钟,以确保完全交联。


PCR后清理(30分钟)
PCR程序结束后小号,取出管从热循环和添加65μ升的SPRI珠(1.3比率μ升的SPRI珠1 μ升PCR产物)。上下吹打混合。
让其在室温下静置5-10分钟。
放在磁力架上几分钟,以使溶液澄清。
除去并丢弃上清液。
保持管在磁性支架,添加200 μ升80%乙醇中。
完全去除并丢弃上清液。
重复小号TEPS我5 -我6。  
执行快速自旋,并用20除去剩余的上清液μ升移液管,从而避免空气通过立即执行下一步骤干燥该珠。
从磁力架上取下,加入22 µl 10 mM Tris-HCl pH 8 ,并全速涡旋。让我们坐了5分钟至1个小时。
放在磁铁架上并使其清除。
用移液器将液体移至新的1.5毫升试管中,避免转移珠子。


Tapestation分析(图3)和DNA测序
使用带有D1000试剂或同等试剂的Agilent 4200 TapeStation ,通过毛细管电泳确定文库的大小分布和浓度。
注:我们使用定量通过Tapestation估算图书馆浓度和每个库稀释至2纳米池基于175-1片段摩尔浓度之前,000 bp的范围。浓度2 nM的公顷小号,已经确定为在所使用的最佳浓度库HiSeq由佛瑞德基因组学的Hutch共享资源。


混合条形码库,以根据需要实现等于表示,瞄准的终浓度如制造商推荐的。混合后,如果需要除去任何残留的PCR引物,请进行SPRI珠纯化。
按照制造商的说明,对带条形码的文库进行双末端Illumina测序。为了获得最大的经济效益,双末端PE25足以映射到大型基因组。
注意:在HiSeq 2通道快速运行流动池上使用双末端25 × 25测序,我们获得了约3亿个总图谱读数,或者当有96个样品混合以获得大致相等的摩尔浓度时,每个样品约有300万个图谱读数。




图3 。Tapestation性质进行低格-数RNAPIIS5P CUTAC实验。Tagmentation物进行20分钟,在37 ℃下在CUTAC -己缓冲器。这些样品的代表性轨迹示于图4A。


数据分析


使用Bowtie2版本2.3.4.3将配对末端的读数与hg19对齐,并具有以下选项:--end-to-end-非常敏感--no- unalal --no-mixed --no-corcordant --phred33 -I 10- X700。为绘制大肠杆菌残留片段,我们也使用--no-overlap --no-dovetail选项以避免实验基因组与残留的大肠杆菌DNA的交叉映射。用于校准。轨迹作为归一化计数的底图文件进行制作,这是每个碱基对上总计数的分数,该分数根据hg19基因组的大小进行缩放。
注意:对于使用同一抗体平行进行的样品,要对一系列样品进行校准,我们使用pA-Tn5携带的大肠杆菌片段数来进行计数,就像普通的插入操作一样。我们在Github中的示例脚本可用于基于刺入或大肠杆菌残留DNA进行校准。


我们的CUT&Tag数据处理和分析教程为CUT&Tag序列数据的映射和分析提供了分步指导。用于大多数数据分析工具芯片起的数据,如BEDT ö醇,皮卡德,和deepTools,可以在使用CUT&标签数据(图3- 5 )。专为CUT&RUN设计分析工具/标签数据包括SEACR峰来电者,也可作为一个公共的网络服务器,并CUT&R UNTool小号。




图4 。可访问的DNA对应于RNA聚合酶II(RNAPII)起始形式的结合位点。一。曲目显示了1号染色体组蛋白基因簇的概况,其中12个小的无内含子基因在所有分裂细胞中高水平表达。尽管RNAPIIS5P CUT&Tag在每个基因上均表现出广泛的富集性,但适用于RNAPIIS5P表位的CUTAC协议(天然(RNAPIIS5P CUTAC-N)或交联(RNAPIIS5P CUTAC-X))产生的启动子轮廓清晰,优于H3K4me2 CUTAC± 1,6-己二醇(青绿色)或最佳的K562 ATAC-seq数据集(绿色),全部都下采样为320万个映射片段。注意在规模RNAPIIS5P CUTAC(0-1500和K4me2-CUTAC / ATAC(0-150)之间的10倍的差异。三个混合谱系白血病细胞系(KOPN8,SEM获得类似的结果,和RS411)和H1胚胎干细胞下降到〜2000细胞,没有变化的协议,用于低格编号进行。括号中的数字以百万映射配对末端的估计库大小的读取。BD 。RNAPIIS5P占据可接近的染色质部位的K562细胞。乙。从左至右:K4me2 CUT&标签,K4me2 CUTAC,RNAPIIS5P CUT和标签,RNAPIIS5P CUTAC,全方位ATAC ,并快速ATAC数据集进行间苗至320万片,并在由ENCODE项目产生的数据使用MACS2名为ATAC-SEQ波峰对准(ATAC ENCODE )。ç 。同(A)不同的是使用仅subnucleosome尺度的片段(≤ 120 bp的)。CUTAC RNAPIIS5P部位是由高品质ATAC-SEQ数据几乎没有区别,直接表明ATAC-S当量映射起始位点RNA聚合酶II。的形式d 。同 (A),仅使用> 120 bp片段。将ENCODE ATAC- seq片段下采样至320万,去除ChrM (线粒体DNA),并使用MACS2调用峰。热图以ENCODE ATAC-seq峰顶为中心,并按占用的5 kb跨度排序。快速ATAC是ATAC-SEQ的改进版本,减少线粒体读取(Corces等人,2016) ,和全方位ATAC是一种改进的版本,额外地改进的信号与噪声的比率(Corces等人,2017)。ATAC_ENCODE是当前的ENCODE标准(Consortium等,2020)。




图5 。RNAPIIS5P CUTAC具有很高的敏感性和特异性。对来自指示的K562数据集的映射片段进行了采样,并使用MACS2调用了峰。甲)峰数(左)和乙)的分数读取为峰CUT&标签(三角形)和CUTAC(正方形)对H3K4me2,RNAPIIS5(起始形式),RNAPIIS2P(伸长率表),RNAPIIS2P / S5P型材,和全方位ATAC (绿色)。用于RNAPIIS5P的CUTAC表现出最佳的灵敏度(低采样时最多的峰)和最佳的信噪比(所有采样水平下的最高FRiP )。Tagmentation是在37 10分钟℃,在CUTAC标签缓冲器。


菜谱


核提取1(NE1)缓冲液
混合1毫升的1M HEPES-KOH pH值7.9,500微升的1M的氯化钾,12.5微升的2M的亚精胺,500微升的10%的Triton X-100,和10毫升的在38毫升卫生署甘油2 O,并添加1罗氏(Roche)Complete Protease Inhibitor不含EDTA。


洗涤缓冲液
混合1毫升的1M的HEPES pH为7.5,将1.5ml的5M的氯化钠,和12.5微升的2M的亚精胺,使最终体积至50ml与卫生署2 O,并添加1罗氏完全蛋白酶抑制剂无EDTA片剂。将缓冲液在4°C下最多保存2天。


绑定缓冲区
混合200微升的1M HEPES-KOH pH值7.9,100微升的1M的氯化钾,10微升的1M的CaCl 2 ,和10微升的1M的MnCl 2 ,和使最终体积至10ml与卫生署2 O.存储缓冲器在4°C长达数月。


抗体缓冲液
混合5微升的200 × BSA用1ml洗涤缓冲液和冰上冷却。BSA存在于某些但不是全部抗体溶液中,该缓冲液中的0.1%BSA有助于防止珠粒在以后的步骤中丢失。


300洗涤缓冲液
混合1毫升的1M的HEPES pH为7.5,将3ml的5M的氯化钠,和12.5微升的2M的亚精胺,使最终体积至50ml与卫生署2 ö ,并添加1罗氏完全蛋白酶抑制剂无EDTA片剂。在4°C下最多保存2天。


CUT&标签Tagmentation缓冲
混合1 ml的300洗涤缓冲液和10 µl的1 M MgCl 2 (至10 mM)。

CUTAC标签缓冲
CUTAC标签:混合197微升的卫生署2 O,2微升的1M的TAPS pH为8.5 ,和1μl的1M的的MgCl 2 (10mM的TAPS和5毫摩尔MgCl 2 )。将缓冲液在4°C下最多保存1天。
CUTAC -己:混合97微升的卫生署2 O,100微升的20%(W / V)的1,6-己二醇,2微升的1M的TAPS pH为8.5 ,和1μl的1M的的MgCl 2 (10mM的TAPS, 5 mM MgCl 2 10%1,6-己二醇)。将缓冲液在4°C下最多保存1天。
TAPS清洗缓冲液
混合1 ml的dH 2 O,10 µl的1 M TAPS pH 8.5、0.4 µl的0.5 M EDTA(10 mM TAPS,0.2 mM EDTA)


0.1%SDS释放溶液
在1 ml的dH 2 O中混合10 µl的10%SDS和10 µl的1 M TAPS pH 8.5


0.67%Triton中和溶液
混合67μl的10%Triton-X100 + 933μldH 2 O


致谢


该协议改编自先前的出版物(Henikoff等人,2020年),并扩展为包括RNAPIIS5P CUTAC,以及可从GEO(GSE158327)获得的数据集。我们感谢特里·布赖森和恭Codomo来样加工,弗雷德双雄基因组学共享资源用于DNA测序,我们实验室的有益讨论的成员,批判地阅读手稿和保罗·塔尔伯特。SH是霍华德·休斯医学研究所的研究员。这项工作得到了霍华德·休斯医学研究所(SH),美国国立卫生研究院的R01 HG010492(SH)和R01 GM108699(KA)的支持以及Chan-Zuckerberg Initiative(SH)的HCA种子网络的资助。


利益争夺


SH已提交与此工作相关的专利申请。JGH和KA宣布没有竞争利益。


参考


Andersson,R.,Sandelin ,A.和Danko ,CG(2015)。转录调控元件的统一体系结构。趋势基因31(8):426-433。
阿诺德,PR,威尔斯,AD和李XC(2019)。增强子RNA在基因表达和细胞命运调控中的多样性和新兴作用。前沿细胞生物学杂志7:377。
奥尔巴赫,RK,Euskirchen的,G.,Rozowsky ,J.,拉马尔文森,N.,Moqtaderi ,Z.,Lefrancois ,P.,Struhl ,K.,格斯坦,M.和Snyder,M。(2009)。使用Sono-Seq映射可访问的染色质区域。PROC国家科科学院科学USA 106(35):14926-14931。
Bownes ,M。(1990)。将P元素优先插入果蝇种系中表达的基因中。分子遗传学222(2-3):457-460。
Buenrostro ,JD,Giresi ,PG,扎巴,LC,昌,HY和绿叶,WJ(2013)。天然染色质易位,可对开放染色质,DNA结合蛋白和核小体位置进行快速而敏感的表观基因组分析。Nat Methods 10(12):1213-1218。
Buenrostro ,JD,Wu,B.,Litzenburger ,UM,Ruff,D.,Gonzales,ML,Snyder,MP,Chang,HY和Greenleaf,WJ(2015)。单细胞染色质的可及性揭示了调节变异的原理。自然523(7561):486-490。
Chereji ,RV,Eriksson,PR,Ocampo,J.,Prajapati,HK和Clark,DJ(2019)。启动子DNA的可及性不是染色质介导的基因调控的主要决定因素。Genome Res 29(12):1985-1995。
Corces ,MR,Buenrostro ,JD,Wu,B.,Greenside,PG,Chan,SM,Koenig,JL,Snyder,MP,Pritchard,JK,Kundaje ,A.,Greenleaf,WJ,Majeti ,R。和Chang,HY (2016)。谱系特异性和单细胞染色质可及性图表显示了人类造血和白血病的进化。Nat Genet 48(10):1193-1203。
Corces ,MR,Trevino,AE,汉密尔顿,EG,Greenside,PG,Sinnott-Armstrong,NA,Vesuna ,S.,Satpathy ,AT,Rubin,AJ,Montine ,KS,Wu,B.,Kathiria ,A.,Cho ,SW,Mumbach ,MR,卡特,AC,Kasowski ,M.,奥尔洛夫,LA,Risca ,VI,Kundaje ,A.,Khavari,PA,Montine ,TJ,绿叶,WJ和Chang,HY(2017)。改进的ATAC-seq协议可减少背景并询问冷冻组织。Nat Methods 14(10):959-962。
克劳福德,通用电气,霍尔特,IE,穆利金,JC,戴维·D,布莱克斯利,R。,博法德·G,扬·A,马西耶洛,C。,格林,ED,沃尔夫斯堡,TG,柯林斯,FS和国家卫生研究院内壁测序,C。(2004年)。通过DNase超敏位点的全基因组恢复鉴定基因调控元件。PROC国家科科学院科学USA 101(4):992-997。
Dorschner ,MO,Hawrylycz ,M.,Humbert,R.,Wallace,JC,Shafer,A.,Kawamoto,J.,Mack,J.,Hall,R.,Goldy,J.,Sabo,PJ,Kohli,A Li,Q.,McArthur,M。和Stamatoyannopoulos ,JA(2004)。通过定量染色质分析对功能元件进行高通量定位。Nat Methods 1(3):219-225。
Giresi ,PG,Kim,J.,McDaniell ,RM,Iyer ,VR和Lieb ,JD(2007)。FAIRE(甲醛辅助调节元素的分离)从人染色质中分离出活性调节元素。Genome Res 17(6):877-885。
Gottschling ,DE(1992)。酿酒酵母中的端粒近端DNA对体内的甲基转移酶活性是难治的。PROC国家科科学院科学USA 89(9):4062-4065。
Henikoff ,S.,Henikoff ,JG,Kaya-Okur,HS和Ahmad,K.(2020年)。高效的染色质可及性通过核小体连接的标签原位作图。Elife 9:e63274。
Jack,RS和Eggert,H。(1990)。限制酶限制了果蝇染色体中DNA序列的访问。EMBO J 9 (8):2603-2609。
Kaya-Okur,HS,Janssens,DH,Henikoff ,JG,Ahmad,K.和Henikoff ,S.(2020年)。使用CUT&Tag进行低成本的高效染色质分析。纳特Protoc 15(10):3264-3283。
Kaya-Okur,HS,Wu,SJ,Codomo ,CA,Pledger,ES,Bryson,TD,Henikoff ,JG,Ahmad,K.和Henikoff ,S.(2019)。CUT&Tag用于小样本和单细胞的有效表观基因组分析。Nat Commun 10(1):1930年。
财团,EP,摩尔,JE,Purcaro ,MJ,普拉特,HE,爱泼斯坦,CB,Shoresh的,N.,阿德里安,J.,Kawli ,T.,戴维斯,CA,Dobin ,A.,考尔,R.,Halow ,J.,Van Nostrand,EL,Freese,P.,Gorkin ,DU等。(2020)。人类和小鼠基因组中DNA元素的扩展百科全书。自然583(7818):699-710。
Oberbeckmann ,E.,沃尔夫,M.,Krietenstein ,N.,苍鹭,M.,Ellins ,JL,施密德,A.,克雷布斯,S.,百隆,H.,盖兰,U。和科伯,P。(2019 )。酿酒酵母基因组的绝对核小体占据图。 Genome Res 29(12):1996-2009。
里夫斯河(1978)。非洲爪蟾卵母细胞的核小体结构扩增了核糖体基因。生物化学17(23):4908-4916。
Weintraub,H.和Groudine ,M.(1976)。活性基因中的染色体亚基具有改变的构象。科学193(4256):848-856。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Henikoff, S., Henikoff, J. G. and Ahmad, K. (2021). Simplified Epigenome Profiling Using Antibody-tethered Tagmentation. Bio-protocol 11(11): e4043. DOI: 10.21769/BioProtoc.4043.
  2. Henikoff, S., Henikoff, J. G., Kaya-Okur, H. S. and Ahmad, K. (2020). Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation.Elife 9: e63274.
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