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May 2020

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ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH
基于ATAC-Seq的哺乳动物细胞染色体外环状DNA鉴定及其反向PCR和FISH验证   

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Abstract

Recent studies from multiple labs including ours have demonstrated the importance of extrachromosomal circular DNA (eccDNA) from yeast to humans (Shibata et al., 2012; Dillon et al., 2015; Møller et al., 2016; Kumar et al., 2017; Turner et al., 2017; Kim et al., 2020). More recently, it has been found that cancer cells obtain a selective advantage by amplifying oncogenes on eccDNA, which drives genomic instability (Wu et al., 2019; Kim et al., 2020). Previously, we have purified circular DNA and enriched the population using rolling circle amplification followed by high-throughput sequencing for the identification of eccDNA based on the unique junctional sequence. However, eccDNA identification by rolling circle amplification is biased toward small circles. Here, we report a rolling circle-independent method to detect eccDNA in human cancer cells. We demonstrate a sensitive and robust step-by-step workflow for finding novel eccDNAs using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) combined with a Circle_finder bioinformatics algorithm to predict the eccDNAs, followed by its validation using two independent methods, inverse PCR and metaphase FISH (Fluorescence in situ Hybridization).

Keywords: Circular DNA (环状DNA), eccDNA (染色体外环状DNA), ATAC-seq (结合高通量测序技术的靶向开放染色质的研究方法), Inverse PCR (反向PCR), FISH (荧光原位杂交)

Background

Extrachromosomal circular DNAs (eccDNAs) are unique DNA molecules that carry genetic information in addition to the chromosomal DNAs. These eccDNAs have been found in different organisms from yeast to humans (Shibata et al., 2012; Dillon et al., 2015; Møller et al., 2016; Kumar et al., 2017; Turner et al., 2017; Kim et al., 2020). The length of eccDNAs ranges from small (less than 1kb, also called microDNAs) to large (megabase-long). While the small eccDNAs with micro homology ends may promote genetic heterogeneity (Shibata et al., 2012) or produce short RNAs if transcribed (Paulsen et al., 2019), the long eccDNAs may harbor complete genes and regulatory elements such as enhancers (Morton et al., 2019; Wu et al., 2019; Koche et al., 2020). Emerging evidence suggests that eccDNAs could play underappreciated roles in regulating gene expression and genome instability, which ultimately contributes to the selective advantage of cells (Gresham et al. 2010, Koo et al., 2018; Hull et al., 2019). In particular, oncogene-carrying eccDNAs are highly amplified in human cancers and correlate with open chromatin, increased oncogene expression, and chromosome structural rearrangement, in addition to being associated with poor outcomes (Wu et al., 2019; Kim et al., 2020; Koche et al., 2020). Uncovering eccDNAs in the circulation also makes them prospective targets for diagnostic purposes (Kumar et al., 2017; Sin et al., 2020).


The growing research on eccDNAs calls for tool development for eccDNA discovery. Historically, eccDNAs of various sizes were detected by karyotyping, electron microscopy, Southern blotting, and 2-D gel electrophoresis (reviewed in Paulsen et al., 2018). More recently, various high-throughput sequencing (HTS) technologies have been exploited to facilitate the discovery of new eccDNAs (Shibata et al., 2012; Møller et al., 2015; Kim et al., 2020). The basic idea for eccDNA detection via HTS methods is based on their distinctive circular feature – eccDNAs of high confidence can be identified from paired-end reads that (1) could not map as inward pairs on the linear genome, and (2) contain the unique circular junctional sequence that represents the chromosome breakage/ligation point. A unique junctional sequence (shown as “E-A” in Figure 1A) that is not present in the normal reference genome could be formed through ligation of the two ends of a linear DNA, thus creating the circular DNA. However, the majority of eccDNA sequencing pipelines utilize multiple displacement amplification (MDA), an efficient method to amplify small amounts of DNA via rolling circle amplification, which would preferentially amplify short circles. Therefore, we sought to develop an MDA-independent pipeline that incorporates several additional validation assays.


Recently, we demonstrated a robust workflow to detect and validate new eccDNAs from human cancer cell cultures (Kumar et al., 2020). Specifically, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and a Circle_finder algorithm was employed for new eccDNA prediction. ATAC-seq, first developed in 2013, utilizes engineered Tn5 transposase to cut open chromatin regions (including eccDNAs that are less chromatinized) and insert transposase-associated adapter DNAs (Buenrostro et al., 2013 and 2015). The Circle-finder algorithm (refer to Software for link access) predicts eccDNAs from paired-end sequencing based on: (1) the presence of split reads (one read maps to two sites in the genome); (2) the two fragments on the split read maps on the same chromosome and same strand; and (3) the continuous read maps between the two fragments on the split read and on the opposite strand to the split read. Predicted eccDNAs can be evaluated by two independent validation assays (Figure 1). Inverse PCR (Figure 1B) will specifically amplify eccDNAs with a primer pair that spans the unique junctional sequence (shown as “E-A”); such a primer pair faces outward on genomic DNA and results in no amplification. Alternatively, eccDNA can be visually confirmed by metaphase FISH (Figure 1C), which can detect both genomic DNA signals overlapping with main chromosomes and signals from eccDNAs that do not overlap with chromosomes.



Figure 1. Overview of eccDNA identification and validation by ATAC-seq, inverse PCR, and metaphase FISH


Materials and Reagents

  1. 50 ml Falcon conical tubes (Fisher Scientific, catalog number: 1443222)

  2. 15 ml Falcon conical tubes (Fisher Scientific, catalog number: 1495949B)

  3. 1.5 ml DNA LoBind tubes (Eppendorf, catalog number: 022431021)

  4. 1 ml pipette tip

  5. 100-200 μl pipette tip

  6. 100 mm Petri dish

  7. 0.2 μm filter

  8. Microscope glass slides (Fisher Scientific, catalog number: 4951F-001)

  9. 22 mm × 50 mm cover glass (Fisher Scientific, catalog number: 12-545E)

  10. Parafilm (Thermo Fisher Scientific, catalog number: S37440)

  11. Aluminum foil (Thermo Fisher Scientific, catalog number: 14-648-236)

  12. Mammalian cells in culture. In this protocol, we used the ovarian cancer cell line, OVCAR8, and the prostate cancer cell line, C4-2B, cultured in RPMI medium (Corning, catalog number: 10-040-CV) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, catalog number: 26140079) and 1% penicillin-streptomycin (Thermo Fisher Scientific, catalog number: 15-140-122)

  13. SybrGold dye (Invitrogen, catalog number: S11494)

  14. 0.5% trypsin-EDTA (Thermo Fisher Scientific, catalog number: 15400054)

  15. UltraPure DNase/RNase-free distilled water (Thermo Fisher Scientific, catalog number: 10977015)

  16. 1 M Tris-HCl, pH 7.5 (Thermo Fisher Scientific, catalog number: 15567027, store at 4°C, shelf life: 6 months)

  17. 5 M NaCl solution (Thermo Fisher Scientific, catalog number: AM9760G, store at room temperature)

  18. 1 M MgCl2 solution (Thermo Fisher Scientific, catalog number: AM9530G, store at room temperature)

  19. Dulbecco’s phosphate-buffered saline or DPBS, no calcium, no magnesium (Thermo Fisher/Gibco, catalog number: 14190144, store at 4°C, shelf life: 36 months)

  20. 10% Nonidet P40 substitute (Millipore/Sigma-Aldrich, catalog number: 11332473001, store at 4°C, keep protected from light, shelf life: 24 months)

  21. 10% (w/v) Tween-20 (Millipore/Sigma-Aldrich, catalog number: 11332465001, store at 4°C under inert gas and keep protected from light, shelf life: 24 months)

  22. 20 mg/ml digitonin in DMSO (Promega, catalog number: G9411, store in -20 °C)

  23. DNA Clean & Concentrator Kit (ZYMO, catalog number: D4033)

  24. Nextera DNA Sample Preparation Kit (Illumina, catalog number: FC-121-1030, store at -20°C)
    Note: This kit has been discontinued and can be purchased separately: Tagmentation DNA Enzyme/TDE (Illumina, catalog number: 15027865) and Tagmentation DNA Buffer/TDB (Illumina, catalog number: 15027866).

  25. Nextera Index Kit, 24 indexes (Illumina, catalog number: 15055289, store at -20°C)

  26. NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs, catalog number: M0541, store at -20°C)

  27. Phosphate-buffered saline, pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023)

  28. Qiagen HiSpeed Plasmid Midi Kit (Qiagen, catalog number: 12643)

  29. Isopropanol (Fisher Chemical, catalog number: A516-500)

  30. Ethanol (Thermo Fisher Scientific, catalog number: A4094)

  31. Glycogen (Thermo Fisher Scientific, catalog number: AM9510)

  32. Plasmid-safe ATP-dependent DNase (Lucigen, catalog number: E3101K)

  33. QIAquick PCR Purification Kit (Qiagen, catalog number: 28104)

  34. KOD Hot-Start DNA Polymerase (Millipore/Sigma-Aldrich, catalog number: 71086)

  35. Thymidine (Millipore/Sigma-Aldrich, catalog number: T1895)

  36. 10 mg/ml KaryoMax Colcemid solution in PBS (Thermo Fisher Scientific, catalog number: 15212012)

  37. Potassium chloride (Millipore/Sigma-Aldrich, catalog number: P9541)

  38. Formamide (Millipore/Sigma-Aldrich, catalog number: 47670)

  39. Sodium chloride (Thermo Fisher Scientific, catalog number: BP358)

  40. Sodium citrate (Millipore/Sigma-Aldrich, catalog number: W302600)

  41. BAC FISH Probe label with 5-fluorescein (Empire Genomics, catalog number: RP11-732I3 and RP11-765O11)

  42. Rubber cement (Elmer’s Rubber Cement, catalog number: EPIE904)

  43. Nonidet P-40 (Sigma, catalog number: I8896)

  44. Dextran sulfate (Thermo Fisher Scientific, catalog number: BP1585)

  45. VectaShield Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200-10)

  46. Nail polish (OPI Nail Lacquer)

  47. Nikon immersion oil for the confocal microscope (Thermo Fisher Scientific, catalog number: 12-624-66A)

  48. 1% (10 mg/ml) digitonin (see Recipes, store at -20°C as aliquotes, stable for 6 months)

  49. ATAC-Resuspension Buffer (see Recipes)

  50. ATAC-Lysis Buffer (see Recipes)

  51. ATAC-Wash Buffer (see Recipes)

  52. ATAC-Reaction Mastermix (see Recipes)

  53. 100 mM thymidine solution (see Recipes)

  54. 75 mM KCl Hypotonic Solution (see Recipes)

  55. Carnoy’s Fixative Solution (see Recipes)

  56. 20× Saline-Sodium-Citrate (SSC) buffer (see Recipes)

  57. Hybridization Buffer (see Recipes)

  58. FISH Denaturation Buffer (see Recipes)

  59. FISH Wash Buffer 1 (see Recipes)

  60. FISH Wash Buffer 2 (see Recipes)

Equipment

  1. Cell culture incubator

  2. Tissue culture hood

  3. Tabletop microcentrifuge (Eppendorf, model: 5424)

  4. Thermomixer (Thermo Scientific, catalog number: 13687711)

  5. PCR machine with heated lid (Eppendorf, model: Mastercycler Pro)

  6. Tabletop centrifuge (Eppendorf, model: 5804)

  7. Water bath (Thermo Fisher Scientific, Isotemp)

  8. Coplin jar (Local Company)

  9. Hybridization chamber (Thermo Fisher Scientific, Isotemp)

  10. Chemical fume hood (Bellco Glass Inc.)

  11. Brightfield microscope (Olympus)

  12. Confocal microscope (Nikon, model: Ti-E eclipse series)

  13. Computer with enough data storage capacity up to TB

Software

  1. Circle_finder (github, https://github.com/pk7zuva/Circle_finder/blob/master/circle_finder-pipeline-bwa-mem-samblaster.sh); pre-requisite installation to run Circle_finder: bedtools (https://github.com/arq5x/bedtools2), samtools (http://samtools.sourceforge.net), parallel (https://www.gnu.org/software/parallel/), bwa (https://github.com/lh3/bwa), samblaster (https://github.com/GregoryFaust/samblaster)

  2. Cutadapt (https://cutadapt.readthedocs.io/en/stable/)

  3. AR Elements Software (Nikon, Japan)

  4. ImageJ (NIH, USA)

Procedure

  1. ATAC-seq from cultured mammalian cells

    1. Nuclei isolation from cultured mammalian cells

      1. Pellet 50,000 mammalian cells in culture into a 1.5 ml DNA LoBind tube.

        Note: Check cell viability prior to the experiment by Trypan Blue staining and ensure cell viability is at least 95%. Please refer to the original ATAC-seq protocol (Corces, 2017) for treatment of cells with DNase to remove extracellular DNAs or to separate cells via ficoll gradient if viability is lower than 95%.

      2. Wash cells in ice-cold DPBS twice at 500 × g.

      3. Add 50 μl ice-cold ATAC-LB to each tube, pipette up and down 3 times with a 100-200 μl pipette tip. Incubate on ice for 3 min.

        Note: We have used a 3-min lysis time with several cell lines, including HCT116, OVCAR8, and C4-2B. The lysis time may need to be increased for specific tough-to-lyse cell lines. The efficiency of cell lysis can be checked by Trypan Blue staining under the microscope (blue staining suggests successful lysis).

      4. Immediately dilute the 50 μl lysate by adding 1 ml ice-cold ATAC-WB into the tube. Invert tube 3 times to mix. Spin at 500 × g for 10 min at 4°C.

      5. Carefully remove the supernatant (containing the cytoplasmic fraction*), first by a 1-ml pipette tip followed by a 100-200-μl pipette tip. The pellet now contains the nuclei, which should be used for the tagmentation reaction immediately.

        *Note: The cytoplasmic fraction can be saved for RNA extraction later.

    2. Transposition/tagmentation reaction and clean-up

      1. Resuspend the nuclei pellet in 50 μl ATAC-RM by pipetting up and down 6 times. Incubate in a thermomixer at 37°C for 30 min with 1,000 RPM constant mixing.

      2. Add 250 μl DNA Binding Buffer (from Zymo DNA Clean and Concentrator Kit) to each 50 μl ATAC reaction, mix well by pipetting up and down 3 times. Transfer 300 μl mixture to each column sitting on a collection tube (both from Zymo DNA Clean and Concentrator Kit), spin down at 15,000 × g for 30 s and discard liquid in the collection tube.

      3. Wash column with 200 μl DNA Wash Buffer (from Zymo DNA Clean and Concentrator Kit), spin down at 15,000 × g for 30 s.

        Note: Make sure ethanol is added to the Wash Buffer according to the manufacturer’s guidelines.

      4. Repeat the wash with 200 μl DNA Wash Buffer. Spin down at 15,000 × g (or maximum speed) for 2 min to ensure efficient removal of all buffers.

      5. Discard the previous collection tube. Carefully displace the column into a new 1.5-ml tube. Add 21 μl ddH2O to the center of the column and spin down at 15,000 × g (or maximum speed) for 30 s to collect eluted DNA. Tagmented DNA can be stored at -20°C (in low DNA-binding tube) at this point if not proceeding to the next step immediately.

    3. DNA library PCR amplification and clean-up

      1. Set up PCR reaction in 0.2-ml PCR tubes on ice:

        20 μl eluted DNA

        2.5 μl 25 μM Nextera i5 primer (from Nextera index kit)

        2.5 μl 25 μM Nextera i7 primer (from Nextera index kit)

        25 μl 2X NEBNext master mix

        Note: If multiple samples will be pooled for sequencing, different combinations of i5 and i7 indexed primers need to be used for each sample. Please refer to specific NGS sequencing platform for recommendation about choice of index combinations. For Illumina sequencing with Nextera index, please refer to Illumina Index Adapters Pooling Guide.

      2. PCR amplification for 10 cycles with the following PCR cycling program (lid set to 105°C):

        Step 1. 72°C, 5 min

        Step 2. 98°C, 30 s

        Step 3. 98°C, 10 s

        Step 4. 63°C, 30 s

        Step 5. 72°C, 1 min

        (Repeat steps 3-5 for a total of 5-10 cycles)

        Step 6. 4°C on hold

        Note: The optimal number of PCR cycle needs to be optimized for each specific cell line to ensure enough final products while avoiding over-amplification. The cycle number can be optimized by testing a range of different cycle numbers (lowest from 5 cycles to ensure sequencing adaptors attached to DNA fragments) using a small portion of the tagmented DNA. The amplification can be monitored by qPCR or run on an agarose gel (Figure 2).



        Figure 2. Example of optimizing ATAC-seq PCR cycle number by a qPCR method. Shown here is an example of using qPCR to determine the optimal number of PCR cycles for ATAC-seq library preparation in C4-2 cells. A separate ATAC reaction with the same number of starting materials was set up in parallel to the actual ATAC-seq reaction, and finished Step A2. The qPCR reaction was set up similar to Step A3a, with the addition of 1:400 dilution of SybrGold Dye (Invitrogen S11494), and set up in a qPCR-compatible plate. The qPCR program was set up the same as Step A3b with real-time fluorescence reading for a total of 20 cycles. Upon finishing qPCR, the Rn (normalized reporter) value is plotted against the cycle number and the saturation point (indicated by the dashed blue line) is determined from the plot. From the Rn-Cycle plot, the cycle number is selected when it reaches 1/3 of the saturation signal (indicated by the red dashed line). In this particular example, a PCR cycle number of 6 was selected. If a separate ATAC reaction is not intended, a small portion (for example, 1/16) of the reaction after Step A2 can be used as input for the qPCR reaction; and the remaining reaction can be used for the final PCR amplification.


      3. Clean up the 50 μl PCR reaction using a ZYMO DNA Clean and Concentrator Kit (similar to above). Elute in 15 μl H2O. Confirm the size distribution of the amplified library using a Bioanalyzer HS DNA Chip (Figure 3).

      4. Submit ATAC-seq libraries for paired-end Illumina sequencing.

    4. Identify potential eccDNA genomic coordinates using the circle_finder algorithm

      1. Remove the adaptor sequence using the cutadapt program (Martin, 2011) with the following parameters: cutadapt -a ADAPT1 -A ADAPT2 -o out1.fastq -p out2.fastq in1.fastq in2.fastq.

        Note: ADAPT1 = CTGTCTCTTATACACATCTCCGAGCCCACGAGAC, ADAPT2 = CTGTCTCTTATACACATCTGACGCTGCCGACGA. in1.fastq and in2.fastq are input fastq files before adaptor removal. out1.fastq and out2.fastq are paired-end fastq files after adaptor removal.

      2. The Circle_finder pipeline first maps the paired-end reads (read length should be >75 bases long) onto the genome (in this case hg38 genome build) using bwa aligner (bwa-mem) (Li, 2013). While mapping paired-end reads to the genome, Circle_finder collects those paired-end reads where one read is mapped in a contiguous manner and the partner read is mapped in a non-contiguous (split-read) manner, supposing one end maps on the body of the circular DNA and the other on the circular DNA ligation junction. Returning to the list of paired-end IDs that mapped uniquely to three sites (one contiguously mapped reads and two-position for reads mapped in a split-read manner) in the genome, the pipeline identifies paired-end IDs where the contiguously mapped read is between the two split reads and on the opposite strand (Figure 1A). The start of the first split read and the end of the second read is annotated as the start and end of the eccDNA. The pipeline (a single script – see next step) to identify eccDNA from paired-end sequencing data of read length >than 75 bases long coming from a specific locus (nonchimeric eccDNA) of any length is available through our GitHub page (refer to software section).

      3. Generate the genome index file: this only needs to be generated once for a specific genome. For example, command to generate an index file for human genome hg38: bwa index hg38.fa.

        Note: hg38.fa is the fasta file for the genome of interest (hg38 in this case). This will generate an index under the same name as the genome fasta file name.

      4. Use the bash script with the following logic: #Usage: bash Script_name “Number of processors” “/path-of-whole-genome-file/hg38.fa” “paired-end fastq file 1” “paired-end fastq file 2” “minNonOverlap between two split reads” “Sample name” “genome build”. An example script is shown here (code as a single line):

        #bash /path-of-script/directory/microDNA-pipeline-bwa-mem-samblaster.sh 16 /path-of-script-directory/hg38.fa S1_R1.fastq S1_R2.fastq 10 S1 hg38

        Note 1: The pipeline takes seven arguments as below.

        Argument 1 = “Number of processors”;

        Argument 2 = “Genome fasta file” for example “hg38.fa”;

        Argument 3 = “paired-end fastq file 1”;

        Argument 4 = “paired-end fastq file 2”;

        Argument 5 = “minNonOverlap between two split reads”, for example 10;

        Argument 6 = “Sample name”, user may choose any name for their sample;

        Argument 7 = “genome build”, user may choose their genome build, such as hg38.

        Note 2: The chance of identifying eccDNA depends on sufficient sequencing depth and read length to cover the eccDNA-specific junctional sequence. Here, we used around 100 million read pairs with a 150-bp read length on the Illumina HiSeq platform.

      5. The output file from “circle_finder” will be a file named “microDNA-JT.txt,” which contains four columns including chromosome number, start position of eccDNA coordinate, end position of eccDNA coordinate, and number of junctional tags.



      Figure 3. Representative ATAC-seq library size distribution. ATAC-seq was performed on OVCAR8 nuclei. A characteristic ladder distribution is detected by the bioanalyzer due to the nucleosome arrangement on chromatin.


  2. Validation of selective eccDNA by inverse PCR

    Note: EccDNA should be treated delicately. EccDNA are prone to shearing and degradation when frozen, vortexed, or kept for long-term storage at a low concentration of DNA.

    1. Culturing and harvesting cells

      1. Harvest 107-108 human cancer cells by trypisinization into a 15-ml tube.

      2. Centrifuge at 300 × g, 4°C for 5 min, remove media by aspiration.

      3. Resuspend cells with 10 ml ice-cold phosphate-buffered saline (PBS).

      4. Centrifuge at 300 × g for 5 min.

      5. Remove PBS by careful aspiration.

      6. Repeat washing steps for a total of two washes, immediately proceed to next step.

    2. EccDNA isolation by plasmid columns

      1. Resuspend cells in 6 ml Buffer P1 from the Qiagen HiSpeed Plasmid Midi Kit.

        Note: Add RNase A solution to Buffer P1 prior to experiment and store at 4°C.

      2. Add 6 ml Buffer P2 from the Qiagen HiSpeed Plasmid Midi Kit and mix by gently inverting 4-6 times.

      3. Incubate on the bench at room temperature for 5 min.

      4. Add 6 ml pre-chilled Buffer P3 from the Qiagen HiSpeed Plasmid Midi Kit and mix by gently inverting 4-6 times.

      5. Set up QIAfilter Cartridge from the Qiagen HiSpeed Plasmid Midi Kit by removing the outlet nozzle cap and sitting the cartridge on top of a waste container (such as a 50-ml conical flask). Add the cell lysate (total of 18 ml) into the QIAfilter Cartridge with the cap attached. Incubate at room temperature for 10 min.

      6. While waiting, set up HiSpeed Midi Tip from the Qiagen Plasmid Midi Kit on top of a waste container (such as a 50-ml conical flask) and equilibrate with 4 ml Buffer QBT.

      7. Remove cap from the QIAfilter Cartridge and filter the cell lysate into the HiSpeed Tip by gently inserting the plunger into the cartridge.

      8. After the cell lysate has passed through the HiSpeed Tip, add 10 ml Buffer QC.

      9. After Buffer QC has passed through the HiSpeed Tip, move it to a clean 50-ml conical tube.

      10. Add 5 ml Buffer QF to elute DNA from the HiSpeed Tip.

      11. Add 3.5 ml isopropanol and mix gently by inversion, incubate at room temperature for 5 min.

      12. Attach the QIAprecipitator Module from the Qiagen HiSpeed Plasmid Midi Kit onto a 20-ml syringe after removing the plunger from the syringe.

      13. Add the DNA-isopropanol solution from Step B2k to the syringe and gently push the solution through the QIAprecipitator Module into a waste container.

      14. Remove the 20-ml syringe from the QIAprecipitator Module, pull out the plunger, re-attach the 20-ml syringe to the QIAprecipitator Mocule, add 20 ml 70% ethanol to the syringe and gently push the plunger to wash the QIAprecipitator Module.

      15. Dry the QIAprecipitator Module by pushing air through the module several times until no more liquid can be pushed out. Dry the QIAprecipitator Module outlet with absorbent paper (such as kimwipes).

      16. Detach the Module from the 20-ml syringe. Attach a new 5-ml syringe (without plunger) to the QIAprecipitator Module.

      17. Add 1 ml Buffer TE from the Qiagen HiSpeed Plasmid Midi Kit to the syringe and push through the QIAprecipitator Module with the plunger into a 1.5-ml Eppendorf tube.

      18. Perform ethanol precipitation: Split the 1000 μl DNA solution to 500 μl between two 1.5-ml Eppendorf tubes. Add 1,000 μl 100% ethanol and 1 μg glycogen to each of the tubes. Mix by pipetting up and down gently. Centrifuge in a tabletop centrifuge at 13,000 rpm for 30 min.

      19. Remove supernatant as much as possible with 1,000 μl and 100 μl pipette tips without dislodging the DNA pellet. The pellet should be visible at the bottom of the tube. Air dry for 5 min.

      20. Resuspend the DNA in each tube in 20 μl Buffer TE from the Qiagen HiSpeed Plasmid Midi Kit and combine to one tube (40 μl volume).

    3. Further enrichment of eccDNAs by DNase digestion to remove linear DNA

      1. To 40 μl DNA, add 128 μl ddH2O, 8 μl 25 mM ATP, 20 μl 10× Reaction Buffer (from the Plasmid-safe ATP-dependent DNase Kit), and 4 μl Plasmid-safe ATP-dependent DNase. Incubate at 37°C overnight (10-12 h).

      2. Purify the DNA using the QIAquick PCR Purification Kit by adding 5 volumes of Buffer PB (1,000 μl) to 1 volume of the digested DNA solution (200 μl) and mix gently by inversion. Add the mixture to a QIAquick Spin Column, centrifuge for 1 min at 17,900 × g.

      3. Discard the flowthrough and wash the QIAquick Spin Column with 750 μl Buffer PE (containing ethanol) by centrifugation for 1 min at 17,900 × g. Repeat the wash step for a total of two washes.

      4. Elute the DNA with 50 μl Buffer EB from the QIAquick PCR Purification Kit.

      5. Repeat digestion and purification steps until the DNA concentration no longer decreases. The concentration begins high (>500 ng/μl) and should decrease to a low level (<20 ng/μl).

        Note: For this protocol, we perform a total of 2 rounds of digestion and purification. To optimize the number of digestion/purification rounds needed for each specific cell line, DNA concentration should be measured by Qubit before and after each round of digestion/purification. The concentration will stop decreasing after a certain number of digestion/purification rounds, indicating that the contaminating linear DNA has been successfully removed.

    4. Inverse PCR to detect specific eccDNA using an outward-directed primer set

      1. Perform PCR with primers that target the junction sequence of the eccDNA using the KOD Hot-Start PCR Kit.

      2. Add 25 μl Xtreme buffer, 10 μl dNTPs (2 μM each), 1.5 μl each primer (10 pmol/μl), 1 μl KOD Xtreme Hot-Start Polymerase, 200 ng purified DNA, and enough ddH2O for the final solution to be 50 μl.

      3. PCR amplification using a PCR machine with heated lid (lid set to 105°C):

        Step 1. 94°C, 2 min

        Step 2. 98°C, 10 s

        Step 3. 68°C, 1 min/kb

        (Repeat Step 2-3 for a total of 30 cycles)

      4. The PCR product can then be visualized on an agarose gel and sequenced by Sanger sequencing with the primers used for PCR amplification (Figure 4).



      Figure 4. Representative eccDNA identified from the ATAC-seq library and validated by inverse PCR in OVCAR8 cells. A. EccDNAs were amplified using inverse PCR primers (shown in blue boxes), gel purified, and validated for the presence of junctional sequences by Sanger sequencing. Example Sanger sequencing results can be found in Figure 3D of the original manuscript (Kumar et al., 2020). B. The table represents the distribution of eccDNA on different chromosomes with coordinates and their expected PCR product size; the numbers represent the different lanes on the gel. C. As a negative control, the same inverse PCR primers were used on purified eccDNAs from U2OS cells (lanes 1-8). As a positive control, inverse PCR primers against mitochondrial DNA were used on eccDNAs (lanes 9-10).


  3. Metaphase spread and FISH detection of specific circular DNAs

    1. Prepare the cells for metaphase spread

      1. Seed 5 × 105 cells in each of five 100 mm Petri dishes 24 h before thymidine block.

        Note: Starting cell number is important to get enough mitotic cells for eccDNA detection.

      2. Add 100 mM thymidine solution to a final concentration of 2 mM to each of the 100 mm plates for 16 h. Release cells for 9 h by replacing regular medium without thymidine solution. Repeat another thymidine block (2 mM final concentration, 16 h) to arrest the cells at the G1-S boundary and release cells for 3 h in regular medium.

      3. Add 0.1 μg/ml final concentration Colcemid for 9 h to collect the mitotic cells.

        Note: Check cells under a microscope to confirm the round shaped mitotic cells.

      4. Gently shake off the floating mitotic cells from the culture dish and collect into a 15-ml Falcon tube by centrifuging at 300 × g for 5 min, and wash the pellet twice with cold PBS.

        Note: Mitotic cells are fragile, so it is very important to use a low speed during centrifugation.

      5. Gently resuspend the pellet in 5 ml 75 mM KCl Hypotonic Solution and incubate at 37°C in a water bath for 30 min. Invert the tube gently every 10 min to ensure the cells are in suspension. Add 1 ml Carnoy’s Fixative Solution to the cell suspension dropwise using a 1,000 μl pipette tip and mix by inverting the tube slowly to keep the metaphase chromosome intact.

      6. Centrifuge cells at 300 × g for 5 min and carefully aspirate most of the KCl solution, leaving about 300 μl. Resuspend cell pellet by gently tapping on the tube.

      7. Fix the resuspended cells by adding 5 ml ice-cold Carnoy’s Fixative Solution dropwise using a 1,000 μl pipette tip and invert the tube slowly to mix the cells.

        Note: It is important to invert the tube slowly to avoid fragmentation of the mitotic chromosomes.

        Note: At this stage, fixed cells can be stored at 4°C for several months.

      8. Centrifuge the cells at 300 × g for 5 min and resuspend the pellet gently in 1-2 ml Carnoy’s Fixative Solution.

      9. Humidify the glass slides, putting on a box at 55°C by slanting at a 45-degree angle, and add several drops of fixative cell suspension from 15-20 cm above the slides.

        Note: It is very important to humidify the glass slides for proper disruption of the nuclear membrane and also to drop the fixative cell suspension from the above-mentioned height for proper spreading of the mitotic chromosomes.

      10. Dry the slides at room temperature away from light and stain with VectaShield Mounting Medium containing DAPI to view the mitotic chromosome spread under a microscope. The dry slides containing the mitotic spreads can be stored at 4°C for several months.

    2. Denaturation of slides containing metaphase DNA

      1. Pre-warm 100 ml FISH Denaturation Buffer at 73°C for 5 min.

      2. Immerse the glass slides containing metaphase spread in a Coplin jar containing pre-warmed FISH Denaturation Buffer for 5 min.

      3. Immerse the slides in a Coplin jar containing 1× PBS, pH 7.4 for 5 min.

      4. Dehydrate the slides serially by immerging the slides in 70%, 85%, and 100% ethanol for 2 min each.

      5. Air dry the slides until all the ethanol has evaporated.

    3. Probe denaturation and hybridization

      Note: It is important to keep the FISH probe protected from light. Aluminium foil is used to cover the slides or hybridization chamber.

      1. Denature the FISH probe in Hybridization Buffer (19 μl Hybridization Buffer + 1 μl labeled probe) at 73°C for 5 min and immediately chill on ice.

      2. Apply the probe mixture onto the previously prepared, air-dried metaphase spread slide and cover with a coverslip. Seal the coverslip with rubber cement and place the slides in the humidified box and hybridize at 37°C in the hybridization chamber overnight. Parafilm is used to ensure sealing of the humidified box.

      3. After hybridization, immerse the slides in 1× PBS and remove the rubber cement and coverslip gently.

      4. Wash the slides in a Coplin jar with pre-warmed FISH Wash Buffer 1 at 73°C for 5 min.

      5. Wash the slides with FISH Wash Buffer 2 at room temperature for 5 min.

      6. Air dry the slides in the dark at room temperature, mount with VectorShield DAPI medium, and seal with nail polish.

    4. Image and data analysis

      Capture the images under a confocal microscope with a 63× oil-immersion objective. Set the laser power to output 40% and acquire the image with the full region of interest (512 × 512) at 300 ms exposure times. For the detection of eccDNA, OVCAR8 (experimental cell line) and C4-2 (negative control cell line where we do not see any extrachromosomal signal) were probed against the eccDNA locus Chr2:238136071-238170279 or Chr10:103457331-103528085 (Figure 5). Potential eccDNA signals (indicated by the red arrows) are located off the main chromosomes, while the chromosomal signals overlap with the main chromosomes and usually appear as doublet signals (indicated by the yellow arrows).



      Figure 5. Validation of eccDNA in OVCAR8 cells by metaphase FISH. Metaphase spread of the chromosomes was carried out and the eccDNAs were identified by FISH. A representative eccDNA locus Chr2:238136071-238170279 (top row – probe 1) or Chr10:103457331-103528085 (bottom row – probe 2) was identified from OVCAR8 ATAC-seq and used for specific BAC probe design. The metaphase spreads from C4-2B cells on the left show no extrachromosomal circular DNA (negative control), while the spreads from OVCAR8 cells on the right confirm the presence of an extrachromosomal eccDNA signal (green: BAC probe, blue: DAPI). The red arrow indicates the eccDNA signals (which can be a singlet or a doublet due to replication of the eccDNA). The yellow arrows mark chromosomal DNA signals (which is usually a doublet but can be a singlet because the signal is seen from only a single chromatid).

Data analysis

  1. The OVCAR8 and C4-2B ATAC-seq data has been deposited in Gene Expression Omnibus (accession: GSE145409). Following the Circle-finder algorithm, the identified eccDNA coordinates output can be found in the Supplementary File under the same GEO accession. Hundreds of potential eccDNAs were identified from this dataset, including small circles of less than 1 kb and large circles of 400 kb encompassing several genes [refer to Figure 3 in the original manuscript (Kumar et al., 2020)].

  2. To validate the potential eccDNAs, PCR-based validation was used for 11 eccDNAs from OVCAR8 and 6 eccDNAs from C4-2B. The primer is designed based on the junctional sequence identified from ATAC-seq and will specifically amplify eccDNA but not the genomic DNA (unless tandem duplication). In total, 13 out of 17 eccDNAs were validated by this method [refer to Figure 3 in the original manuscript (Kumar et al., 2020)].

  3. FISH on metaphase spreads can be used to visually validate the presence of selective eccDNAs, but preferentially with large circles. Here, we were able to detect FISH signals off chromosomes corresponding to 34-kb and 71-kb eccDNAs in OVCAR8 cells (Figure 5). To understand the general distribution of eccDNA signals in a cell population, we counted eccDNA FISH signals in more than 20 cells and found a distribution between 0 and 4 eccDNA FISH signals in each cell examined [refer to Figure 4 in the original manuscript (Kumar et al., 2020)].

Recipes

  1. 1% (10 mg/ml) Digitonin

    40 μl 20 mg/ml digitonin stock solution in DMSO

    40 μl ddH2O

    Store at -20°C as aliquots, stable for 6 months

  2. ATAC-Resuspension Buffer (ATAC-RSB) (50 ml)

    500 μl 1 M Tris-HCl, pH 7.4 (10 mM final concentration)

    100 μl 5 M NaCl (10 mM final concentration)

    150 μl 1 M MgCl2 (3 mM final concentration)

    49.25 ml ddH2O

  3. ATAC-Lysis Buffer (ATAC-LB) (500 μl for 8 reactions)

    5 μl 10% Nonidet P40 substitute (0.1% final concentration)

    5 μl 10% Tween-20 (0.1% final concentration)

    5 μl 1% digitonin (0.01% final concentration)

    485 μl ATAC-RSB

    Prepare fresh, keep on ice

  4. ATAC-Wash Buffer (ATAC-WB) (10 ml for 8 reactions)

    100 μl 10% Tween-20 (0.1% final concentration)

    9.9 ml ATAC-RSB

    Prepare fresh, keep on ice

  5. ATAC-Reaction Mastermix (ATAC-RM) (450 μl for 8 reactions)

    225 μl 2× TDB buffer (from Nextera kit, 25 μl per reaction)

    22.5 μl TDE transposase (from Nextera kit, 2.5 μl per reaction)

    148.5 μl DPBS

    4.5 μl 1% Digitonin (0.01% final concentration)

    4.5 μl 10% Tween-20 (0.1% final concentration)

    45 μl H2O

    Prepare fresh, keep on ice

  6. 100 mM Thymidine Solution

    Dissolve 242 mg thymidine powder in 10 ml cell culture grade water and filter through a 0.2-μm filter

  7. 75 mM KCl Hypotonic Solution

    Dissolve 559 mg KCl in 100 ml cell culture grade water and filter through a 0.2-μm filter

  8. Carnoy’s Fixative Solution

    Add 75 ml methanol to 25 ml glacial acetic acid (v/v) to make 100 ml fixative solution

    Prepare under a chemical fume hood

  9. 20× SSC Buffer

    Mix 87.5 g NaCl and 44.1 g sodium citrate in 400 ml water and adjust the pH with a few drops of 12 N hydrochloric acid to pH 7.0

    Adjust the volume with water to 500 ml and filter through a 0.2-μm filter

  10. Hybridization Buffer

    10 ml 20× SSC buffer pH 7.0

    50 ml 100% formamide solution

    10 g dextran sulfate

    Adjust volume to 100 ml with water

    Prepare under chemical fume hood

  11. FISH Denaturation Buffer

    70 ml 100% formamide solution

    10 ml 20× SSC buffer

    20 ml sterile water

    Prepare under chemical fume hood

  12. FISH Wash Buffer 1

    2 ml 20× SSC buffer

    3 ml 10% NP-40

    95 ml water

  13. FISH Wash Buffer 2

    10 ml 20× SSC buffer

    1 ml 10% NP-40

    89 ml water

Acknowledgments

This work was supported by R01 CA060499 and P30 CA044579 to AD and Cancer Training Grant support from T32 CA009109 (PI: Amy Bouton) to TP. We thank all members of the Dutta Lab for many helpful discussions.

Competing interests

The authors declare no competing interests.

References

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简介

[摘要]包括我们在内的多个实验室的最新研究已经证明了酵母染色体外循环DNA(eccDNA)对人类的重要性(Shibata等人,2012;Dillon等人,2015年;米øLeller等人,2016年;Kumar等人,2017年;Turner等人,2017年;Kim等人,2020年)。最近,发现癌细胞通过扩增eccDNA上的癌基因获得选择性优势,从而导致基因组不稳定(Wu等人,2019;Kim等人,2020年)。以前,我们已经纯化了环状DNA,并利用滚动圈扩增和高通量测序来富集群体,用于基于独特连接序列的eccDNA鉴定。然而,用滚圈扩增法鉴定eccDNA的方法偏重于小圆。在这里,我们报道了一种检测人类癌细胞eccDNA的非滚动圆方法。我们展示了一个敏感而稳健的一步一步的工作流程,使用ATAC-seq(转座酶可访问染色质测序分析)结合Circle-finder生物信息学算法预测ECCDNA,然后使用两种独立的方法进行验证,反向PCR和中期FISH(荧光原位杂交)。


[背景]染色体外环状DNA(eccdna)是一种独特的DNA分子,除了携带染色体DNA外,还携带遗传信息。从酵母到人类的不同生物体中都发现了这些eccdna(Shibata等人,2012;Dillon等人,2015年;米øLeller等人,2016年;Kumar等人,2017年;Turner等人,2017年;Kim等人,2020年)。eccdna的长度范围很小(小于

1kb,也称为微DNA)到大(兆碱基长)。虽然具有微同源末端的小eccdna可能促进遗传异质性(Shibata等人,2012)或在转录后产生短rna(Paulsen等人,2019),但长eccdna可能含有完整的基因和调控元件,如增强子(Morton等人,2019;Wu等人,2019年;Koche等人,2020年)。新的证据表明,eccdna可能在调节基因表达和基因组不稳定性方面发挥未被充分认识的作用,这最终有助于细胞的选择性优势(Gresham等人,2010年,Koo等人,2018年;赫尔等人,2019年)。特别是,携带癌基因的eccdna在人类癌症中高度扩增,除了与不良预后相关外,还与染色质开放、癌基因表达增加和染色体结构重排相关(Wu等人,2019;Kim等人,2020年;Koche等人,2020年)。发现循环中的eccdna也使其成为诊断目的的预期靶点(Kumar等人,2017;Sin等人,2020年)。

随着对eccDNA研究的不断深入,eccDNA发现工具的开发势在必行。历史上,通过核型分析、电子显微镜、Southern印迹和2-D凝胶电泳检测到不同大小的eccdna(见Paulsen et al.,2018)。最近,各种高通量测序(HTS)技术被用来促进新的ECCDNA的发现(Shibata等人,2012;米øLeller等人,2015年;Kim等人,2020年)。通过HTS方法检测eccDNA的基本思想是基于其独特的循环特征——从成对末端读取可以识别出高置信度的eccDNA,即(1)不能在线性基因组上映射为内向对,以及(2)包含表示染色体断裂/连接点的独特循环连接序列。正常参考基因组中不存在的独特连接序列(如图1A中的“E-A”)可以通过连接线性DNA的两端形成,从而形成环状DNA。然而,大多数eccDNA测序管道采用多重置换扩增(MDA)技术,这是一种通过滚圈扩增法扩增少量DNA的有效方法,它优先扩增短圈。因此,我们试图开发一个独立于MDA的管道,其中包含了一些额外的验证分析。

最近,我们展示了一个从人类癌细胞培养物中检测和验证新eccdna的强大工作流程(Kumar等人,2020)。具体而言,ATAC-seq(转座酶可及染色质测序分析)和Circle-finder算法被用于新的eccDNA预测。

ATAC-seq于2013年首次开发,利用工程化的Tn5转座酶切割染色质区域

(包括染色质较少的eccdna)和插入转座酶相关的适配器dna(Buenrostro等人,2013和2015)。Circle finder算法(指用于链接访问的软件)通过成对末端测序预测eccdna,其依据是:(1)存在分裂读取(一个读取映射到基因组中的两个位点)(2) 分裂后的两个片段在同一条染色体和同一条链上读取图谱;以及(3)分割读取上的两个片段之间的连续读取映射和分割读取上的相反链上的连续读取映射。预测的eccdna可以通过两个独立的验证分析进行评估(图1)。反向PCR(图1B)将用一对横跨独特连接序列的引物(如“E-a”)特异性地扩增eccdna;这样的一对引物面向基因组DNA,不会导致扩增。或者,中期FISH可以直观地确认eccDNA(图1C),它可以检测与主染色体重叠的基因组DNA信号和与染色体不重叠的eccDNA信号。





图1。ATAC-seq、反向PCR和中期FISH在eccDNA鉴定和验证中的应用

关键字:环状DNA, 染色体外环状DNA, 结合高通量测序技术的靶向开放染色质的研究方法, 反向PCR, 荧光原位杂交


材料和试剂

1.  50 ml Falcon锥形管(Fisher Scientific,目录号:1443222)

2.  15 ml Falcon锥形管(Fisher Scientific,目录号:1495949B)

3.  1.5 ml DNA LoBind试管(Eppendorf,目录号:022431021)

4.  1毫升移液管头

5.  100-200 μl移液管头

6.  100 mm培养皿

7.  0.2μm过滤器

8.  显微镜载玻片(Fisher Scientific,目录号:4951F-001)

9.  22毫米× 50 mm盖玻璃(Fisher Scientific,目录号:12-545E)

10.  Parafilm(赛默飞世尔科技公司,目录号:S37440)

11.  铝箔(赛默飞世尔科技,目录号:14-648-236)

12.  哺乳动物细胞的培养。在该方案中,我们使用卵巢癌细胞系OVCAR8和前列腺癌细胞系C4-2B,在RPMI培养基(康宁,目录号:10-040-CV)中培养,并添加10%胎牛血清(赛默飞世尔科技,目录号:26140079)和1%青霉素链霉素(赛默飞世尔科技,目录号:15-140-122)

13.  SybrGold染料(Invitrogen,目录号:S11494)

14.  0.5%胰蛋白酶EDTA(赛默飞世尔科技,目录号:15400054)

15.  超纯DNase/RNase游离蒸馏水(赛默飞世尔科技,目录号:10977015)

16.  1 M Tris-HCl,pH 7.5(赛默飞世尔科技公司,目录号:15567027,储存于4°C、 保质期:6个月)

17.  5 M NaCl溶液(Thermo Fisher Scientific,目录号:AM9760G,室温储存)

18.  1 M MgCl2溶液(赛默飞世尔科技公司,目录号:AM9530G,室温储存)

19.  Dulbecco磷酸盐缓冲盐水或DPBS,不含钙,不含镁(Thermo Fisher/Gibco,目录号:14190144,储存于4°C、 保质期:36个月)

20.  10%Nonidet P40替代品(密理博/Sigma-Aldrich,目录号:11332473001,储存于4°C、 避光,保质期:24个月)

21.  10%(w/v)吐温-20(密理博/Sigma-Aldrich,目录号:11332465001,储存于4°C在惰性气体下保存,避光,保质期:24个月)

22.  20 mg/ml二甲基亚砜中的洋地黄素(Promega,目录号:G9411,储存于-20°(三)

23.  DNA清洁浓缩试剂盒(ZYMO,目录号:D4033)

24.  Nextera DNA样品制备试剂盒(Illumina,目录号:FC-121-1030,储存于-20°C) 注:此试剂盒已停产,可单独购买:标记DNA

酶/TDE(Illumina,目录号:15027865)和标记DNA缓冲液/TDB(Illumina,目录号:15027866)。

25.  Nextera索引工具包,24个索引(Illumina,目录号:15055289,储存于-20°(三)

26.  NEBNext高保真2× PCR主混合物(新英格兰生物实验室,目录号:M0541,储存于-20°(三)

27.  磷酸盐缓冲盐水,pH 7.4(赛默飞世尔科技,目录号:10010023)

28.  Qiagen HiSpeed质粒Midi试剂盒(Qiagen,目录号:12643)

29.  异丙醇(费希尔化学公司,目录号:A516-500)

30.  乙醇(赛默飞世尔科技公司,目录号:A4094)

31.  糖原(Thermo Fisher Scientific,目录号:AM9510)

32.  质粒安全ATP依赖性DNA酶(Lucigen,目录号:E3101K)

33.  QIAquick PCR纯化试剂盒(Qiagen,目录号:28104)

34.  KOD热启动DNA聚合酶(Millipore/Sigma-Aldrich,目录号:71086)

35.  胸苷(Millipore/Sigma-Aldrich,目录号:T1895)

36.  PBS中的10 mg/ml KaryoMax Colcemid溶液(赛默飞世尔科技,目录号:15212012)

37.  氯化钾(Millipore/Sigma-Aldrich,目录号:P9541)

38.  甲酰胺(Millipore/Sigma-Aldrich,目录号:47670)

39.  氯化钠(赛默飞世尔科技,目录号:BP358)

40.  柠檬酸钠(密理博/Sigma-Aldrich,目录号:W302600)

41.  带有5-荧光素的BAC FISH探针标签(帝国基因组学,目录号:RP11-732I3和RP11-765O11)

42.  橡胶水泥(埃尔默橡胶水泥,目录号:EPIE904)

43.  Nonidet P-40(西格玛,目录号:I8896)

44.  硫酸葡聚糖(赛默飞世尔科学公司,目录号:BP1585)

45.  带DAPI的VectaShield安装介质(Vector Laboratories,目录号:H-1200-10)

46.  指甲油(OPI指甲油)

47.  尼康共聚焦显微镜浸油(赛默飞世尔科学公司,目录号:12-624-66A)

48.  1%(10 mg/ml)洋地黄素(见配方,储存于-20°C作为aliquotes,稳定6个月)

49.  ATAC再悬浮缓冲液(见配方)

50.  ATAC裂解缓冲液(见配方)

51.  ATAC洗涤缓冲液(见配方)

52.  ATAC反应母料(见配方)

53.  100 mM胸苷溶液(见配方)

54.  75 mM KCl低渗溶液(见配方)

55.  卡诺氏固定液(见配方)

56.  20× 生理盐水柠檬酸钠(SSC)缓冲液(见配方)

57.  杂交缓冲液(见配方)

58.  鱼变性缓冲液(见配方)

59.  鱼洗缓冲液1(见配方)

60.  鱼洗缓冲液2(见配方)


设备

1.  细胞培养箱

2.  组织培养罩

3.  台式微量离心机(Eppendorf,型号:5424)

4.  热混合器(Thermo Scientific,目录号:13687711)

5.  带加热盖的PCR机(Eppendorf,型号:Mastercycler Pro)

6.  台式离心机(Eppendorf,型号:5804)

7.  水浴(Thermo Fisher Scientific,Isotemp)

8.  Coplin jar(当地公司)

9.  杂交室(Thermo Fisher Scientific,Isotemp)

10.  化学通风柜(贝尔科玻璃公司)

11.  布莱特菲尔德显微镜(奥林巴斯)

12.  共焦显微镜(尼康,型号:Ti-E eclipse系列)

13.  数据存储容量高达TB的计算机


软件

1.  圆查找器(github,https://github.com/pk7zuva/Circle_finder/blob/master/circle_finder-pipeline-bwa-mem-samblas 三、上海);运行Circle\u finder的先决条件安装:bedtools

(https://github.com/arq5x/bedtools2),samtools公司(http://samtools.sourceforge.net),平行

(https://www.gnu.org/software/parallel/),bwa公司(https://github.com/lh3/bwa),samblaster公司(https://github.com/GregoryFaust/samblaster)

2.  剪切适应(https://cutadapt.readthedocs.io/en/stable/)

3.  AR Elements软件(尼康,日本)

4.  ImageJ(美国国立卫生研究院)


程序

答。培养哺乳动物细胞的ATAC序列

1.  从培养的哺乳动物细胞中分离细胞核

答。将培养中的50000个哺乳动物细胞放入1.5 ml DNA LoBind管中。

注:实验前用台盼蓝染色检查细胞活力,细胞活力至少为95%。请参考原始的ATAC-seq方案(Corces,2017),用DNase处理细胞以去除细胞外DNA,或者如果存活率低于95%,则通过ficoll梯度分离细胞。

b。在500℃下,在冰凉的DPBS中清洗电池两次× g。

c。加50μl将ATAC-LB冷冻至每根管,用100-200移液管上下移液3次μl移液管尖端。在冰上孵育3分钟。

注:我们对几个细胞系使用了3分钟的裂解时间,包括HCT116、OVCAR8和C4-2B。对于特定的难溶细胞系,可能需要延长裂解时间。台盼蓝染色可在显微镜下检查细胞裂解效率(蓝色染色表明裂解成功)。

d。立即稀释50μl在试管中加入1毫升冰镇ATAC-WB进行裂解。将试管倒置3次混合。以500转× g,4分钟时持续10分钟°C。

e。先用1毫升移液管头,然后用100-200毫升移液管小心地除去上清液(含有细胞质部分*)-μl移液管尖端。现在小球中含有原子核,应立即用于标记反应。

*注:细胞质部分可以保存,以便以后提取RNA。

2.  换位/标记反应和清除

答。在50分钟内重新悬浮核弹μ上下移液6次。在温度为37℃的热混合器中培养°C在1000转/分的恒定搅拌下搅拌30分钟。

b。加250μl DNA结合缓冲液(来自Zymo DNA清洁和浓缩试剂盒)各50μl ATAC反应,上下移液3次搅拌均匀。转移300μ在收集管(来自Zymo DNA Clean和浓缩器试剂盒)上的每一个柱中加入混合物,转速为15000× g持续30 s,并丢弃收集管中的液体。

c。带200的洗涤柱μl DNA洗涤缓冲液(来自Zymo DNA清洁和浓缩试剂盒),转速降到15000× g持续30秒。

注意:确保按照制造商的指南将乙醇添加到清洗缓冲液中。

d。重复200次清洗μDNA清洗缓冲液。转速降到15000× g(或最大速度)2分钟,以确保有效拆除所有缓冲器。

e。丢弃先前的收集管。小心地将色谱柱移入1.5毫升的新试管中。加21μl ddH2O到柱中心,在15000转下× g(或最大速度)30 s,以收集洗脱的DNA。标记的DNA可以储存在-20°C(在低DNA结合管中)此时如果不立即进行下一步。

三。DNA文库PCR扩增与纯化

答。在冰上的0.2毫升PCR试管中设置PCR反应:

20μ我洗提了DNA

2.5μ我25岁μM Nextera i5引物(来自Nextera索引工具包)

2.5μ我25岁μM Nextera i7引物(来自Nextera索引工具包)

25μl 2X NEB下一主混音

注:如果将多个样本合并用于测序,则需要对每个样本使用不同的i5和i7索引引物组合。关于指数组合的选择建议,请参考具体的NGS测序平台。有关使用Nextera索引进行Illumina测序的信息,请参阅Illumina索引适配器池指南。

b。用以下PCR循环程序进行10个循环的PCR扩增(lid设置为105°C) 地址:

第一步。72°C、 5分钟

第二步。98°C、 30秒

第三步。98°C、 10秒

第四步。63°C、 30秒

第五步。72°C、 1分钟

(重复步骤3-5,共5-10个循环)

第六步。4°C暂停

注:每个特定细胞系的最佳PCR周期数需要优化,以确保有足够的最终产物,同时避免过度扩增。循环数可以通过使用标记DNA的一小部分测试一系列不同的循环数(最低5个循环,以确保序列适配器连接到DNA片段)来优化。扩增可通过qPCR监测或在琼脂糖凝胶上运行(图2)。



图2。用qPCR方法优化ATAC-seq-PCR循环数的实例。这里显示的是一个使用qPCR来确定C4-2细胞中ATAC-seq文库制备的最佳PCR周期数的示例。与实际的ATAC-seq反应平行地建立具有相同起始材料数量的单独ATAC反应,并且完成步骤A2。qPCR反应的建立与步骤A3a相似,添加1:400稀释的SybrGold染料(Invitrogen S11494),并在qPCR兼容板中建立。qPCR程序的设置与步骤A3b相同,具有总共20个周期的实时荧光读数。完成qPCR后,根据循环数绘制Rn(标准化报告器)值,并根据该图确定饱和点(由蓝色虚线表示)。从Rn循环图中,当循环数达到饱和信号的1/3(由红色虚线表示)时,选择循环数。在该具体实施例中,选择PCR循环数6。如果不打算进行单独的ATAC反应,则步骤A2之后的反应的一小部分(例如,1/16)可以用作qPCR反应的输入;剩余反应可用于最终PCR扩增。


c。清理50μl使用ZYMO DNA清洁浓缩试剂盒进行PCR反应(同上)。在15分钟内洗脱μl水。使用生物分析仪HS DNA芯片确认扩增文库的大小分布(图3)。

d。提交ATAC序列库以进行配对末端测序。

4用圆搜索算法识别潜在的eccDNA基因组坐标

答。使用具有以下参数的cutadapt程序(Martin,2011)删除适配器序列:cutadapt-a ADAPT1-a ADAPT2-o out1.fastq-p out2.fastq in1.fastq in2.fastq。

注:ADAPT1=CTGTCTTATACACATCTCCGAGCCAGAAC,ADAPT2=

CTGTCTTATACACTGACGCTGCCGACGA公司。in1.fastq和in2.fastq是在移除适配器之前输入的fastq文件。取出适配器后,out1.fastq和out2.fastq是成对的端部fastq文件。

b。Circle_finder管道首先使用bwa aligner(bwa mem)将成对末端读取(读取长度应大于75碱基长)映射到基因组(在本例中为hg38基因组构建)(Li,2013)。当将成对末端读取映射到基因组时,Circle\ U finder收集那些成对末端读取,其中一个读取以连续方式映射,而伙伴读取以非连续(拆分读取)方式映射,假设一端映射到环状DNA的主体上,另一端映射到环状DNA连接连接处。返回到唯一映射到基因组中三个位点(一个连续映射的读取和两个位置,用于以拆分读取方式映射的读取)的成对末端id的列表,管道识别成对末端id,其中连续映射的读取位于两个拆分读取之间和相反链上(图1A)。第一次拆分读取的开始和第二次读取的结束被注释为eccDNA的开始和结束。我们的GitHub页面提供了一个管道(一个脚本-见下一步),用于从任何长度的特定位点(非嵌合体eccDNA)读取长度大于75碱基的成对末端测序数据中识别eccDNA(请参阅软件部分)。

c。生成基因组索引文件:对于特定的基因组,只需生成一次。例如,为人类基因组hg38生成索引文件的命令:bwa index hg38.fa。

注:hg38.fa是感兴趣的基因组的fasta文件(本例中为hg38)。这将以与文件名相同的名称生成索引。

d。按以下逻辑使用bash脚本:#用法:bash脚本#name“Number of processors”“/path of whole genome file/hg38.fa”“paired end fastq file 1”“paired end fastq

文件2“minNonOverlap between two split reads”“Sample name”“genome build”。此处显示了一个示例脚本(代码为单行):

#bash/path of script/directory/microDNA-pipeline-bwa-mem-samblaster.sh 16/path of script directory/hg38.fa S1\u R1.fastq S1\u R2.fastq 10 S1 hg38注1:管道包含以下七个参数。

参数1=“处理器数量”;

参数2=“文件”例如“hg38.fa”;

参数3=“成对结束fastq文件1”;

参数4=“成对结束fastq文件2”;

参数5=“两次拆分读取之间的MinNoOverlap”,例如10;

参数6=“样本名称”,用户可以为其样本选择任何名称;

参数7=“基因组构建”,用户可以选择自己的基因组构建,如hg38。

注2:识别eccDNA的机会取决于足够的测序深度和读取长度,以覆盖eccDNA特异性连接序列。在Illumina HiSeq平台上,我们使用了大约1亿个读取对,读取长度为150 bp。
e。“circle\u finder”的输出文件将是一个名为“microDNA JT.txt”的文件,其中包含染色体编号、eccDNA坐标的起始位置、eccDNA坐标的结束位置和连接标签的数量四列。



图3。代表性的ATAC序列库大小分布。对卵母细胞8核进行ATAC-seq。由于核小体在染色质上的排列,生物分析仪检测到一种典型的梯形分布。


B。反向PCR法验证选择性eccDNA

注意:EccDNA应小心处理。EccDNA在低浓度下冷冻、涡流或长期保存时容易发生剪切和降解。

1.  培养和收获细胞

答。用胰蛋白酶法将107-108个人癌细胞收集到15ml试管中。

b。300℃离心× g、 4个°C持续5分钟,通过抽吸除去介质。

c。用10ml冰磷酸盐缓冲盐水(PBS)复苏细胞。

d。300℃离心× 5分钟。

e。小心抽吸去除PBS。

f。重复洗涤步骤共两次,立即进行下一步。

2.  质粒柱分离EccDNA

答。从Qiagen HiSpeed质粒Midi试剂盒将细胞重新悬浮在6 ml缓冲液P1中。

注:实验前向缓冲液P1中加入核糖核酸酶A溶液,保存于4°C。

b。从Qiagen HiSpeed质粒Midi试剂盒中加入6 ml缓冲液P2,轻轻反转4-6次混合。

c。在工作台上室温孵育5分钟。

d。从Qiagen HiSpeed质粒Midi试剂盒中加入6毫升预冷缓冲液P3,轻轻反转4-6次混合。

e。从Qiagen HiSpeed Plasmid Midi工具包中设置Qiagen过滤器滤筒,方法是取下出口喷嘴盖,将滤筒放在废物容器(如50 ml锥形烧瓶)顶部。将细胞裂解液(总共18毫升)加入带盖的滤筒中。在室温下培养10分钟。

f。等待时,将HiSpeed Midi探针从Qiagen质粒Midi试剂盒中置于废物容器(如50 ml锥形瓶)顶部,并用4 ml缓冲液QBT平衡。

g。从滤筒上取下盖子,轻轻地将柱塞插入滤筒中,将细胞裂解物过滤到HiSpeed尖端。

h。待细胞裂解液通过HiSpeed尖端后,加入10mL缓冲液QC。

一。缓冲液QC通过HiSpeed尖端后,将其移到干净的50 ml锥形管中。

j。加入5毫升缓冲液QF从HiSpeed尖端洗脱DNA。

k。加入3.5毫升异丙醇,倒置轻轻搅拌,室温下孵育5分钟。

l。从注射器中取出柱塞后,将Qiagen HiSpeed质粒Midi试剂盒中的沉淀器模块连接到20 ml注射器上。

m。将步骤B2k中的DNA异丙醇溶液添加到注射器中,并轻轻地将溶液通过沉淀器模块推入废物容器中。

n。从QIA沉淀器模块上取下20毫升注射器,拔出柱塞,将20毫升注射器重新连接到QIA沉淀器模块上,向注射器中加入20毫升70%乙醇,轻轻推动柱塞以清洗QIA沉淀器模块。

o。通过多次将空气推入除尘器模块,干燥除尘器模块,直到不再有液体排出。用吸水纸(如kimwipes)擦干除尘器模块出口。

第。从20毫升注射器上取下模块。将一个新的5毫升注射器(不带柱塞)连接到除尘器模块上。

问。从Qiagen HiSpeed质粒Midi试剂盒中加入1 ml缓冲液TE至注射器中,并用柱塞将Qiagen沉淀器模块推入1.5 ml Eppendorf管中。

r。进行乙醇沉淀:将1000μ把DNA溶液稀释到500μl在两个1.5毫升的Eppendorf试管之间。加1000μl 100%乙醇和1μg糖原到每根管子。用移液管轻轻上下混合。在台式离心机中以13000 rpm离心30分钟。

s。尽可能多地用1000去除上清液μl和100μl移液管尖端不移动DNA颗粒。颗粒应该在管的底部可见。风干5分钟。

t。在20分钟内将每个试管中的DNA重新悬浮μl从Qiagen HiSpeed质粒Midi试剂盒中缓冲TE,并合并到一个试管(40μl体积)。

3.  DNA酶切去除线状DNA进一步富集eccdna

答。到40μDNA,加128μl ddH2O,8μl 25毫米ATP,20毫米μ长10× 反应缓冲液(来自质粒安全的ATP依赖性DNA酶试剂盒)和4μ质粒安全ATP依赖性DNA酶。

37℃孵育°C过夜(10-12小时)。


b。使用快速PCR纯化试剂盒,通过添加5体积的缓冲液PB纯化DNA

(1,000 μl) 稀释至1体积的消化DNA溶液(200μl) 轻轻地倒转。将混合物加入快速旋转柱中,在17900下离心1分钟× g。

c。丢弃流通管,用750清洗快速旋转柱μl在17900下离心1分钟,缓冲PE(含乙醇)× g。重复清洗步骤,共清洗两次。

d。用50%洗脱DNAμ从快速PCR纯化试剂盒中提取EB缓冲液。

e。重复消化和纯化步骤,直到DNA浓度不再降低。

浓度开始高(>500ng/μl) 应降低至低水平(<20 ng/μl) 是的。注:本方案共进行2轮消化纯化。为了优化每个特定细胞系所需的消化/纯化轮数,应在每轮消化/纯化前后用量子位测量DNA浓度。经过一定数量的消化/纯化循环后,浓度将停止下降,表明污染线性DNA已被成功去除。

4.  反向PCR检测特异性eccDNA的研究

答。使用KOD热启动PCR试剂盒对靶向eccDNA连接序列的引物进行PCR。

b。加25μ超级缓冲器,10μdNTPs(2个μM(每个),1.5μl每个底漆(10 pmol/μl) ,1个μl KOD Xtreme热启动聚合酶,200 ng纯化DNA,足够的ddH2O使最终溶液为50μl。

c。使用带加热盖的PCR机进行PCR扩增(盖设置为105°C) 地址:

第一步。94°C、 2分钟

第二步。98°C、 10秒

第三步。68°C、 1分钟/kb

(重复步骤2-3,共30个循环)

d。然后可以在琼脂糖凝胶上观察PCR产物,并用用于PCR扩增的引物进行Sanger测序(图4)。



图4。从ATAC-seq文库中鉴定具有代表性的eccDNA,并通过反向PCR在OVCAR8细胞中进行验证。答。eccdna用反向PCR引物扩增(蓝色框中显示),凝胶纯化,并通过Sanger测序验证连接序列的存在。Sanger测序结果示例可在原始手稿的图3D中找到(Kumar等人,2020)。B。该表用坐标表示了eccDNA在不同染色体上的分布及其预期PCR产物大小;数字代表凝胶上的不同通道。C。作为阴性对照,用同样的反向PCR引物从U2OS细胞中纯化eccdna(lane1-8)。作为阳性对照,在eccdna上使用针对线粒体DNA的反向PCR引物(车道9-10)。


C。特异性环状dna的中期扩散和FISH检测

1准备细胞进行中期扩散

答。种子5× 胸腺嘧啶核苷阻断前24小时,在5个100 mm培养皿中各放置105个细胞。

注:起始细胞数对获得足够的有丝分裂细胞进行eccDNA检测很重要。

b。向每个100 mM平板中添加100 mM胸腺嘧啶核苷溶液至最终浓度2 mM,持续16 h。用不含胸腺嘧啶核苷溶液的常规培养基代替,释放细胞9小时。重复另一次胸腺嘧啶核苷阻滞(终浓度2 mM,16 h),使细胞停留在G1-S边界,并在常规培养基中释放细胞3 h。

c。添加0.1μg/ml终浓度秋水仙碱9小时收集有丝分裂细胞。

注:显微镜下检查细胞,确认为圆形有丝分裂细胞。

d。轻轻摇掉培养皿中漂浮的有丝分裂细胞,在300℃下离心,收集到15ml的Falcon管中× g保持5分钟,并用冷PBS洗涤颗粒两次。注意:有丝分裂细胞是脆弱的,所以在离心过程中使用低速是非常重要的。

e。在5 ml 75 mM KCl低渗溶液中轻轻地重新悬浮颗粒,并在37℃下培养°C在水浴中放置30分钟。每10分钟轻轻翻转一次试管,以确保细胞处于悬浮状态。用1000毫升的试剂滴加1毫升卡诺固定液到细胞悬液中μl移液管尖端,缓慢翻转试管混合,以保持中期染色体完整。

f。在300℃下离心分离细胞× g保持5分钟,并小心地吸取大部分KCl溶液,留下约300μl。轻轻敲击试管,使细胞颗粒重新悬浮。

g。用1000毫升的试剂滴加5毫升冰镇卡诺固定液,固定再悬浮的细胞μl移液管尖端并缓慢翻转试管以混合细胞。

注意:缓慢翻转试管以避免有丝分裂染色体断裂是很重要的。

注:在这个阶段,固定单元可以存储在4°好几个月了。

h。在300℃下离心分离细胞× g静置5分钟,然后在1-2 ml卡诺氏固定液中轻轻地重新悬浮颗粒。

一。把载玻片加湿,放在55的盒子里°C倾斜45度,从载玻片上方15-20厘米处滴入几滴固定细胞悬液。

注:湿化玻片对核膜的适当破坏非常重要,从上述高度滴下固定细胞悬液对有丝分裂染色体的适当扩散也非常重要。

j。室温避光干燥玻片,用含DAPI的VectaShield贴装培养基染色,显微镜下观察有丝分裂染色体的扩散。含有有丝分裂扩散的干载玻片可在4℃保存°好几个月了。

2.  含中期DNA载玻片的变性

答。在73℃下预热100毫升鱼变性缓冲液°C持续5分钟。

b。将含有中间相的玻片浸入含有预热鱼变性缓冲液的Coplin罐中5分钟。

c。将载玻片浸入一个装有1× PBS,pH7.4,5分钟。

d。将载玻片分别浸入70%、85%和100%乙醇中2分钟,连续脱水。

e。将载玻片风干,直到乙醇全部蒸发。

3.  探针变性和杂交

注意:保持FISH探头不受光线照射很重要。铝箔用于覆盖载玻片或杂交室。

答。在杂交缓冲液(19)中变性FISH探针μl缓冲液+1μl标记探针)73°冷却5分钟,然后立即在冰上冷却。

b。将探针混合物涂抹在预先制备的风干中期铺展玻片上,并用盖玻片盖住。用橡胶胶密封盖玻片,将玻片放入加湿盒中,并在37℃下混合°在杂交室过夜。加湿箱的密封采用Parafilm。

c。杂交后,将载玻片浸入1× PBS,并轻轻取出橡胶粘合剂和盖玻片。

d。将载玻片放入Coplin罐中,在73℃下用预热的鱼类清洗缓冲液1清洗°C持续5分钟。

e。在室温下用鱼肉洗涤缓冲液2洗涤载玻片5分钟。

f。载玻片在室温下于黑暗中风干,用VectorShield DAPI介质安装,并用指甲油密封。

4.  图像和数据分析

在共焦显微镜下用63× 油浸物镜。将激光功率设置为输出40%,并获取具有完整感兴趣区域(512)的图像× 512)暴露时间为300 ms。为了检测eccDNA,我们检测了OVCAR8(实验细胞系)和C4-2(阴性对照细胞系,我们没有看到任何染色体外信号)的eccDNA位点Chr2:238136071-238170279或

Chr10:103457331-103528085(图5)。潜在的eccDNA信号(红色箭头所示)位于主染色体之外,而染色体信号与主染色体重叠,通常表现为双峰信号(黄色箭头所示)。



图5。中期FISH法检测卵巢癌细胞eccDNA的研究。染色体进行中期扩散,FISH鉴定eccdna。代表性eccDNA位点Chr2:238136071-238170279(顶行–探针1)或

Chr10:103457331-103528085(底行-探针2)从OVCAR8 ATAC序列中鉴定,并用于特定的BAC探针设计。左侧C4-2B细胞的中期扩散未显示染色体外环状DNA(阴性对照),而右侧OVCAR8细胞的扩散证实存在染色体外eccDNA信号(绿色:BAC探针,蓝色:DAPI)。红色箭头表示eccDNA信号(由于eccDNA的复制,可以是单重态或双重态)。黄色箭头标记染色体DNA信号(通常是双峰,但也可以是单峰,因为信号只能从单个染色单体看到)。


数据分析

1.  OVCAR8和C4-2B ATAC-seq数据已保存在基因表达综合数据库中(登录号:GSE145409)。根据圆查找算法,可以在同一地理位置下的补充文件中找到已识别的eccDNA坐标输出。从这个数据集中发现了数百个潜在的eccdna,包括小于1kb的小圆圈和包含多个基因的400kb的大圆圈[参考原始手稿中的图3(Kumar等人,2020)]。

2.  为了验证潜在的eccdna,对来自OVCAR8的11个eccdna和来自C4-2B的6个eccdna进行了基于PCR的验证。引物是根据ATAC-seq的连接序列设计的,可以特异性地扩增eccDNA而不是基因组DNA(除非串联重复)。总的来说,17个eccdna中有13个通过这种方法进行了验证[参考原始手稿中的图3(Kumar等人,2020)]。

3.  中期扩散上的FISH可用于直观地验证选择性eccdna的存在,但优先使用大圆圈。在这里,我们能够在OVCAR8细胞中检测到与34 kb和71 kb ECCDNA相对应的染色体上的FISH信号(图5)。为了了解eccDNA信号在细胞群中的一般分布,我们对20多个细胞中的eccDNA-FISH信号进行了计数,发现每个受检细胞中有0到4个eccDNA-FISH信号的分布[参考原始手稿中的图4(Kumar等人,2020)]。


食谱

1.  1%(10mg/ml)洋地黄素

40μl 20 mg/ml二甲基亚砜中的洋地黄素储备液

40μl ddH2O

存储在-20°C等分,稳定6个月

2.  ATAC再悬浮缓冲液(ATAC-RSB)(50毫升)

500μl 1 M Tris-HCl,pH 7.4(10 mM最终浓度)

100μl 5 M NaCl(10 mM最终浓度)

150μl 1 M MgCl2(3 mM最终浓度)

49.25毫升ddH2O

3.  ATAC裂解缓冲液(ATAC-LB)(500μl代表8个反应)

5μl 10%Nonidet P40替代品(0.1%最终浓度)

5μl 10%吐温-20(0.1%最终浓度)

5μl 1%洋地黄素(0.01%终浓度)

485μl ATAC-RSB公司

准备新鲜的,放在冰上

4.  ATAC洗涤缓冲液(ATAC-WB)(10毫升用于8个反应)

100μl 10%吐温-20(0.1%最终浓度)

9.9毫升ATAC-RSB

准备新鲜的,放在冰上

5.  ATAC反应母料(ATAC-RM)(450μl代表8个反应)

225μl 2级× TDB缓冲液(来自Nextera kit,25μl每反应)

22.5μl TDE转座酶(来自Nextera试剂盒,2.5μl每反应)

148.5μl DPBS公司

4.5μl 1%洋地黄素(0.01%终浓度)

4.5μl 10%吐温-20(0.1%最终浓度)

45μl水

准备新鲜的,放在冰上

6.  100 mM胸苷溶液

将242 mg胸腺嘧啶核苷粉末溶解于10 ml细胞培养级水中,并通过0.2%的滤池过滤-μm过滤器

7.  75mmkcl低渗溶液

将559 mg KCl溶解于100 ml细胞培养级水中,并通过0.2%的滤池过滤-μm过滤器

8.  卡诺固定液

在25 ml冰醋酸(v/v)中加入75 ml甲醇,在化学通风柜下制备100 ml固定液

9.  20× SSC缓冲器

在400 ml水中混合87.5 g NaCl和44.1 g柠檬酸钠,并用几滴12 N盐酸将pH调节至7.0

用水将体积调节至500 ml,并通过0.2%的过滤器过滤-μm过滤器

10.  杂交缓冲液

10毫升20× SSC缓冲液pH 7.0

50毫升100%甲酰胺溶液

10 g硫酸葡聚糖

用水将体积调节到100毫升

在化学通风柜下准备

11.  鱼变性缓冲液

70毫升100%甲酰胺溶液

10毫升20× SSC缓冲器

20毫升无菌水

在化学通风柜下准备

12.  洗鱼缓冲液1

2  20毫升× SSC缓冲器

3  10%NP-40毫升

95毫升水

13.  鱼洗缓冲液2

10毫升20× SSC缓冲器

1毫升10%NP-40

89毫升水


致谢

这项工作得到了R01 CA060499和P30 CA044579对AD和T32 CA009109(PI:Amy Bouton)对TP癌症培训补助金的支持。我们感谢杜塔实验室的所有成员进行了许多有益的讨论。

相互竞争的利益相互竞争的利益


作者声明没有相互竞争的利益。


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Su, Z., Saha, S., Paulsen, T., Kumar, P. and Dutta, A. (2021). ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH. Bio-protocol 11(9): e4003. DOI: 10.21769/BioProtoc.4003.
  2. Kumar, P., Kiran, S., Saha, S., Su, Z., Paulsen, T., Chatrath, A., Shibata, Y., Shibata, E. and Dutta, A. (2020). ATAC-seq identifies thousands of extrachromosomal circular DNA in cancer and cell lines. Sci Adv 6(20): eaba2489.
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