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Low-input Capture-C: A Chromosome Conformation Capture Assay to Analyze Chromatin Architecture in Small Numbers of Cells
低样品量Capture-C:采用染色体构象采集分析法分析少量细胞中的染色质结构   

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Abstract

Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This protocol describes two new low-input Capture-C approaches that can generate high-quality 3C interaction profiles from 10,000-20,000 cells, depending on the resolution used for analysis.

Keywords: Nuclear organization(核结构), Chromatin architecture(染色质构型), Chromosome conformation capture (3C)(染色体构象捕获(3C)), Capture-C(Capture-C), Low input(低样品量), Small cell numbers(少量细胞)

Background

3C techniques play a key role in investigating how nuclear organization and structural interactions between regulatory elements relate to gene activity (Dekker et al., 2002). As these interactions are highly tissue-specific, it is crucial that 3C experiments are performed in well defined, purified cell populations.

A major limitation of 3C techniques is the large numbers of cells required: current methods use between 100,000 and 10 million cells (Davies et al., 2017). Many primary tissues and rare cell populations are not available in these numbers. We have therefore developed two new low-input Capture-C approaches that can generate high-quality interaction profiles from ~20,000 cells at maximum resolution (individual DpnII fragments), and from ~10,000 cells using windowing based analysis (Oudelaar et al., 2017).

Materials and Reagents

  1. 3C library preparation
    1. LowBind DNA 1.5 ml microtube
    2. Phase Lock Gel (PLG) Light tubes (VWR, Quantabio, catalog number: 733-2477 )
    3. Genomic DNA ScreenTape (Agilent Technologies, catalog number: 5067-5366 )
    4. Tapestation Loading Tips (Agilent Technologies, catalog number: 5067-5153 )
    5. 1 M glycine (Sigma-Aldrich, catalog number: G7126 )
    6. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
    7. Growth media
    8. 37% formaldehyde (Sigma-Aldrich, catalog number: 252549 )
    9. Ethanol absolute > 99.8% (VWR, catalog number: 20821.330 , or equivalent)
    10. Dry ice or liquid nitrogen
    11. PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9932 )
    12. SDS (20% v/v in water) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9820 )
    13. Triton X-100 (20% v/v in water) (Sigma-Aldrich, catalog number: T8787 )
    14. DpnII (50,000 U/ml) (New England Biolabs, catalog number: R0543M )
    15. T4 DNA ligase (30 U/μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EL0013 )
    16. Proteinase K (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0491 )
    17. RNase (DNase-free) (Roche Diagnostics, catalog number: 11119915001 )
    18. Phenol-chloroform-isoamyl alcohol (PCI) 25:24:1 (Sigma-Aldrich, catalog number: 77617 )
    19. 3 M NaOAc (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9740 )
    20. GlycoBlue (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9515 )
    21. Genomic DNA Reagents (Agilent Technologies, catalog number: 5067-5365 )
    22. TaqMan Universal PCR Master Mix II without UNG (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4440040 )
    23. KAPA Sybr Fast Universal (Kapa Biosystems, catalog number: KK4602 )
    24. Fresh lysis buffer (see Recipes)
      1. Tris pH 8 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9855G )
      2. Sodium chloride (NaCl) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9760G )
      3. Igepal CA-630 (100 μl of 10%) (Sigma-Aldrich, catalog number: I8896 )
      4. cOmplete Protease Inhibitor Cocktail (1 tablet in 2 ml of PCR grade water for 25x; store at -20 °C) (Sigma-Aldrich, Roche Diagnostics, catalog number: 11873580001 )
      5. PCR grade water
    25. qPCR primers (Appendix I)

  2. Addition of Illumina sequencing adaptors by ligation or tagmentation to generate Capture-C libraries
    LI-Capture-C
    1. Covaris microTUBE AFA Fiber pre-split snap-cap 6 x 16 mm (Covaris, catalog number: 520045 )
    2. LowBind DNA 1.5 ml microtube
    3. PCR tube
    4. D1000 ScreenTape (Agilent Technologies, catalog number: 5067-5582 )
    5. TapeStation Loading Tips (Agilent Technologies, catalog number: 5067-5153 )
    6. NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, catalog number: E7370S/L )
    7. Agencourt Ampure XP SPRI Beads (Beckman Coulter, catalog number: A63881 )
    8. NEBNext Multiplex Oligos for Illumina Primer set 1 (New England Biolabs, catalog number: E7335S/L )
    9. NEBNext Multiplex Oligos for Illumina Primer set 2 (New England Biolabs, catalog number: E7500S/L )
    10. Herculase II Fusion Polymerase Kit (Agilent Technologies, catalog number: 600677 )
    11. PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9932 )
    12. Ethanol absolute > 99.8% (VWR, catalog number: 20821.330 , or equivalent)
    13. D1000 Reagents (Agilent Technologies, catalog number: 5067-5583 )
    14. Qubit dsDNA BR Assay kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32850 )

    Tag-Capture-C
    1. LowBind DNA 1.5 ml microtube
    2. PCR tube
    3. High Sensitivity D1000 ScreenTape (Agilent Technologies, catalog number: 5067-5584 )
    4. D1000 ScreenTape (Agilent Technologies, catalog number: 5067-5582 )
    5. TapeStation Loading Tips (Agilent Technologies, catalog number: 5067-5153 )
    6. Nextera DNA Sample Preparation Kit (Illumina, catalog number: FC-121-1030 / 1031 )
    7. Zymo DNA Clean & Concentrator-5 Kit (Zymo Research, catalog number: D4013 )
    8. KAPA HiFi PCR Kit with dNTP (KK2102) (Roche Diagnostics, catalog number: 07958846001 )
    9. Custom designed index primers
    10. Agencourt Ampure XP SPRI Beads (Beckman Coulter, catalog number: A63881 )
    11. PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9932 )
    12. Ethanol absolute > 99.8% (VWR, catalog number: 20821.330 , or equivalent)
    13. High Sensitivity D1000 Reagents (Agilent Technologies, catalog number: 5067-5585 )
    14. D1000 Reagents (Agilent Technologies, catalog number: 5067-5583 )
    15. Qubit dsDNA BR Assay kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32850 )

  3. Enrichment for viewpoints of interest by oligonucleotide capture
    1. Safeseal Microcentrifuge Tubes (Sorenson BioScience, catalog number: 39640T )
    2. D1000 ScreenTape (Agilent Technologies, catalog number: 5067-5582 )
    3. Tapestation Loading Tips (Agilent Technologies, catalog number: 5067-5153 )
    4. Nimblegen SeqCap EZ Hybridization and wash kit (Roche Diagnostics, catalog number: 05634261001 )
    5. Nimblegen SeqCap EZ Accessory kit v2 (Roche Diagnostics, catalog number: 07145594001 )
    6. 1 μg/μl COT DNA of relevant species (Mouse) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18440016 )
    7. M-270 Streptavidin Dynabeads (Thermo Fisher Scientific, InvitrogenTM, catalog number: 65305 )
    8. Agencourt Ampure XP SPRI Beads (Beckman Coulter, catalog number: A63881 )
    9. PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9932 )
    10. Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32850 )
    11. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32851 )
    12. D1000 Reagents (Agilent Technologies, catalog number: 5067-5583 )
    13. KAPA Library Quantification Complete Kit (Universal) (Kapa Biosystems, catalog number: KK4824 )
    14. LI-Capture-C
      Nimblegen SeqCap EZ HE-oligo kit A (Roche Diagnostics, catalog number: 06777287001 )
      Nimblegen SeqCap EZ HE-oligo kit B (Roche Diagnostics, catalog number: 06777317001 )
    15. Tag-Capture-C
      xGen Custom Blocking Oligos (P5/i5 & P7/i7 Nextera blocker)

Equipment

  1. Centrifuge
  2. Incubator (any)
  3. Eppendorf Thermomixer C (or equivalent shaking incubator) (Eppendorf, model: ThermoMixer® C , catalog number: 5382000015)
  4. P10, P20, P200 and P1000 pipettes (any)
  5. Thermocycler (any)
  6. NanoDrop
  7. Agilent 2200 TapeStation (Agilent Technologies, model: Agilent 2200 TapeStation System )
    Note: Assessments on the Agilent TapeStation can also be performed on the Agilent BioAnalyzer.
  8. Sonicator (Covaris, model: S220 Focused-ultrasonicator)
  9. DynaMag-2 (Thermo Fisher Scientific, catalog number: 12321D )
  10. Vacuum Concentrator (SciQuip, CHRIST series)

Procedure

Overview
Note: Overview of the procedure (Graphics adapted from Davies et al., 2017).


Steps

  1. 3C library preparation
    1. Fixation (2 h)
      Note: The fixation procedure described below has been optimized for erythroid cells. Depending on the cell type used, some of centrifugation steps might need to be adjusted to ensure proper pelleting.
      1. Pre-cool centrifuge to 4 °C. Chill glycine, PBS, and lysis buffer (see Recipes).
      2. Collect cells from tissue and make single-cell suspensions of 10,000 to 106 cells in 1 ml growth media. If sorting cells, sort into 100 μl media and add media afterwards to a total volume of 1 ml.
      3. Add 54 μl 37% formaldehyde (2% final concentration), mix well and incubate for 10 min at RT on a rocking or tumbling incubator.
      4. Quench by adding 150 μl 1 M cold glycine (1/8 of the final volume. i.e., 1.2 ml).
      5. Centrifuge for 15 min/500 x g/4 °C.
      6. Carefully remove supernatant, do not disturb pellet. Can leave ~5% behind.
      7. Wash pellet by gently resuspending in 1 ml cold PBS.
      8. Centrifuge for 15 min/500 x g/4 °C.
      9. Carefully remove supernatant, do not disturb pellet. Can leave ~5% behind.
      10. Resuspend pellet in 500 μl cold lysis buffer.
      11. Incubate for 20 min on ice.
      12. Snap freeze with ethanol and dry ice or liquid nitrogen. Snap freezing aids digestion so cells can be thawed again for digestion at this point or stored long-term at -80 °C.
        SAFE STOPPING POINT: Store at -80 °C.
    2. Digestion (1 day)
      1. Pre-warm a ThermoMixer to 37 °C.
      2. Prepare Digestion Mix (make a master mix for multiple libraries):
        PCR grade water
        152.5 μl
        10x DpnII Buffer
        20 μl
        20% SDS
        2.5 μl
      3. Defrost aliquots of 10,000 to 106 formaldehyde-fixed cells for each reaction.
      4. Centrifuge for 10 min/15,000 x g/4 °C.
      5. Carefully remove all the lysis buffer, using a P20 to remove residual buffer without disturbing the pellet.
      6. Resuspend in 175 μl of Digestion Mix.
      7. Shake horizontally (1,400 rpm) for 1 h at 37 °C using the ThermoMixer.
      8. Add 16 μl of 20% Triton X-100 (1.7% final concentration) and shake for 1 h at 37 °C.
      9. Add first aliquot of 3 μl of DpnII enzyme and shake at 37 °C for at least two hours.
      10. Add a second aliquot of 3 μl of DpnII enzyme at the end of the day and shake overnight.
      11. Add a further 3 μl of DpnII enzyme the next morning and shake for at least three more hours.
    3. Ligation (1 day)
      1. Place the digests on the 65 °C block for 20 min to heat inactivate the restriction enzyme.
      2. Cool the digest on ice and cool the ThermoMixer to 16 °C.
      3. Make Ligation Mix (make a master mix for multiple libraries):
         PCR grade water
        125 μl
        10x ligation buffer
        36 μl
      4. Add 161 μl Ligation Mix to digest.
      5. Add 4 μl T4 ligase and shake at 300 rpm/16 °C for ~22 h.
    4. De-crosslinking (1 day)
      Add 2 μl Proteinase K (20 mg/ml) and incubate at 65 °C overnight.
    5. DNA extraction (1 day)
      1. Add 4 μl of RNase to the ligation reaction and incubate at 37 °C for 30 min.
      2. Add 350 μl phenol-chloroform-isoamyl alcohol and vortex thoroughly.
      3. Transfer to a 2 ml PLG Light tube and spin for 5 min/15,000 x g/RT.
      4. Transfer the upper layer, ~350 μl, to a new tube.
      5. Add 35 μl of 3 M NaOAc and 2 μl of GlycoBlue and mix by inversion.
      6. Add 960 μl of 100% ethanol and mix by inversion.
      7. Freeze at -20 °C overnight.
        SAFE STOPPING POINT: DNA should be precipitated for at least one night; this can be extended to several days without affecting recovery.
      8. Pre-cool centrifuge to 4 °C during incubation.
      9. Centrifuge for 45 min/21,000 x g/4 °C and carefully discard liquid.
      10. Wash pellet with 1 ml 70% ice-cold ethanol.
      11. Spin for 10 min/21,000 x g/4 °C.
      12. Remove ethanol and repeat ethanol wash (steps A5j-A5k).
      13. Remove ethanol, spin in a microfuge and use a pipette to remove residual ethanol.
        Dry at room temperature and resuspend pellet. When proceeding with LI-Capture-C, resuspend in 124 μl water. For Tag-Capture-C, the amount of water depends on the input, as several tagmentation reactions are set up in parallel (explained below). Use 20 μl water per multitude of 15,000 cells and round up (e.g., 30,000 cells:40 μl water; 50,000 cells:80 μl water).
        SAFE STOPPING POINT: Store at -20 °C.
    6. Quantification and QC (3 h)
      Note: The NanoDrop or similar spectrophotometers are unreliable for quantifying 3C libraries due to residual DTT from the ligation buffer.
      1. Assess 1 μl of 3C library using the Agilent Genomic ScreenTape system to determine the size distribution of library (Appendix I).
      2. For LI-Capture-C–take 3 μl of 3C library and 27 μl water to make a 1/10 dilution; for Tag-Capture-C–take 1 μl of 3C library and 29 μl water to make a 1/30 dilution.
        Perform qPCR for digestion efficiency, using the dilution. Genomic DNA can be used as the undigested control (Appendix I). Digestion efficiency should be > 75%.

  2. Addition of Illumina sequencing adaptors by ligation or tagmentation to generate Capture-C libraries
    LI-Capture-C
    –Based on the protocol for the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)
    Notes:
    1. In this step, it is important to maintain library complexity by maximizing input DNA and minimizing DNA losses during the reactions and cleanups. When more than 1 μg of DNA is available, perform parallel preparations. These can be pooled during cleanups and at the end.
    2. The NEB protocol often recommends removing the DNA from the beads with a few μl more water than is necessary for the next step in the protocol. One can avoid doing this to minimize losses, but this means being very careful with the bead cleanups as contamination by the beads or ethanol can inhibit the following reaction.
    3. We use the Covaris S220 Focused Ultrasonicator for sonication. If using a different model sonicator, use high molecular weight gDNA to optimize sonication for a modal distribution around 200 bp in size (Appendix II).
    4. The Illumina indices that you put on to the library need to be complementary to the Nimblegen HE blocking oligonucleotides required in the capture step (the kits do not match; Appendix II). Buying both primer set 1 and primer set 2 eliminates waste of the more expensive Nimblegen blocking kit.
    5. No size selection is necessary as adaptor dimers will not be captured. All Ampure XP bead cleanups are performed with 1.8x volumes of beads.

    1. Sonication (1 h)
      1. Take 120 μl of 3C library (up to 1 μg) and transfer to a Covaris microtube. Avoid making bubbles.
      2. Shear DNA to 200 bp with the following settings:
        Duty cycle: 10%
        Intensity: 5
        Cycles per burst: 200
        Time: 360 sec
        Set mode: Frequency sweeping
      3. Ampure XP SPRI bead cleanup:
        1. Transfer reaction from the Covaris microtube to a lowBind DNA 1.5 ml microtube.
        2. Add 220 μl beads, pipette up and down 10 times, allow to bind at RT for 5 min.
        3. Place on the DynaMag, discard liquid when clear.
        4. Add 500 μl of fresh 80% ethanol without removing from the DynaMag. Avoid disturbing beads by running the ethanol down the front of the tube. Allow to sit on the rack for 30 sec at RT, then remove ethanol.
        5. Remove ethanol and add another 500 μl of fresh 80% ethanol, allow to sit for 30 sec at RT and remove all the ethanol.
        6. Spin down on microfuge and remove residual ethanol with a P10 pipette.
        7. Air dry at room temperature on the DynaMag until matt in appearance.
          Note: Be careful not to over dry the beads as this will result in increased DNA losses.
        8. Resuspend beads in 56 μl water to elute DNA, mix by pipetting 10 times.
        9. Incubate at RT for 2 min.
        10. Recover 55.5 μl for End Repair reaction.
      4. Optional: Assess 1 μl of sonicated material using the Agilent D1000 (Regular or High Sensitivity, depending on your input) ScreenTape system (Appendix II).
      SAFE STOPPING POINT: Store at -20 °C.
    2. End repair and adaptor ligation (2 h)
      1. Combine 55.5 μl of DNA (5 ng-1 μg), 6.5 μl 10x End Repair Reaction Buffer (Green) and 3 μl End Prep Enzyme Mix (Green). Mix by pipetting and briefly spin down.
      2. Incubate in a thermocycler:
        20 °C for 30 min
        65 °C for 30 min
      3. Add 15 μl Blunt/TA Ligase Master Mix (Red), 2.5 μl NEBNext Adaptor* (Red), 1 μl Ligation Enhancer (Red). Mix by pipetting and briefly spin down.
        *Note: If DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina (provided at 15 μM) 10-fold in 10 mM Tris-HCl or 10 mM Tris-HCl with 10 mM NaCl to a final concentration of 1.5 μM, use immediately.
      4. Incubate for 15 min at 20 °C in a thermocycler.
      5. Add 3 μl of USER Enzyme (Red). Mix by pipetting.
      6. Incubate for 15 min at 37 °C in a thermocycler.
      7. Ampure XP SPRI bead cleanup:
        1. Transfer reaction to a lowBind DNA 1.5 ml microtube.
        2. Add 160 μl beads, pipette up and down 10 times, allow to bind at RT for 5 min.
        3. Place on the DynaMag, discard liquid when clear.
        4. Add 500 μl of fresh 80% ethanol without removing from the DynaMag. Avoid disturbing beads by running the ethanol down the front of the tube. Allow to sit on the rack for 30 sec at RT, then remove ethanol.
        5. Remove ethanol and add another 500 μl of fresh 80% ethanol, allow to sit for 30 sec at RT and remove all the ethanol.
        6. Spin down in a microfuge and remove residual ethanol with a P10 pipette.
        7. Air dry at room temperature on the DynaMag until matt in appearance.
          Note: Be careful not to over dry the beads as this will result in increased DNA losses.
        8. Resuspend beads in 29 μl water, mix by pipetting 10 times.
        9. Incubate at RT for 2 min.
        10. Transfer 28.5 μl eluted DNA to a PCR tube.
    3. PCR addition of indices (2 h)
      Note: Carefully choose your index to allow for multiplexing using the Illumina pooling guidelines.
      1. Perform PCR as described below.


      2. Ampure XP SPRI bead cleanup:
        1. Transfer reaction to a lowBind DNA 1.5 ml microtube.
        2. Add 90 μl beads, pipette up and down 10 times, allow to bind at RT for 5 min.
        3. Place on the DynaMag, discard liquid when clear.
        4. Add 500 μl of fresh 80% ethanol without removing from the DynaMag. Avoid disturbing beads by running the ethanol down the front of the tube. Allow to sit on the rack for 30 sec at RT, then remove ethanol.
        5. Remove ethanol and add another 500 μl of fresh 80% ethanol, allow to sit for 30 sec at RT and remove all the ethanol.
        6. Spin down in a microfuge and remove residual ethanol with a P10 pipette.
        7. Air dry at room temperature on the DynaMag until matt in appearance.
          Note: Be careful not to over dry the beads as this will result in increased DNA losses.
        8. Resuspend beads in 31 μl water, mix by pipetting 10 times.
        9. Incubate at RT for 2 min.
        10. Recover 30 μl.
      3. Assess 1 μl of material using the Agilent D1000 ScreenTape system (Appendix II page 3).
      4. Quantify library using Qubit dsDNA BR assay kit.
      SAFE STOPPING POINT: Store at -20 °C.

    Tag-Capture-C
    Notes:
    1. In this step, it is important to maintain library complexity by maximizing input DNA and minimizing DNA losses during the reactions and cleanups. The maximum input for a tagmentation reaction is 50 ng. When input is higher, run several separate reactions in parallel; these can be pooled after the cleanup.
    2. We use custom designed index primers, for which the sequences can be found in the Appendix. We order these as TruGrade Primers from IDT.
    3. No size selection is necessary as adaptor dimers won’t be captured. All Ampure XP bead cleanups are performed with 1.8x volumes of beads.

    1. Tagmentation (1 day)
      1. Prepare tagmentation reactions with reagents from the Nextera DNA Sample Preparation Kit in a PCR tube:
        < 50 ng 3C library in 20 μl water
        25 μl TD Buffer
        5 μl of TD Enzyme
      2. Mix by pipetting, followed by a quick spin.
      3. Incubate at 55 °C for 12-24 h to ensure all material is tagmented.
      4. Zymo spin column cleanup:
        1. Label tubes and add 180 μl of Zymo DNA Binding Buffer.
        2. Add 50 μl of tagmented DNA. Gently pipette up and down 10 times to mix.
        3. Transfer sample mixture to a Zymo spin column in a collection tube.
        4. Centrifuge for 30 sec at 15,000 x g. Discard the flow-through.
        5. Add 200 μl Zymo DNA Wash Buffer to the column. Centrifuge for 30 sec at 15,000 x g.
        6. Repeat step B1d.v (Tag-Capture-C section) and centrifuge in addition 60 sec at 15,000 x g.
        7. Transfer the column to a 1.5 ml tube and add 34.5 μl of water to the column matrix. Incubate at RT for 2 min.
        8. Centrifuge for 1 min at 15,000 x g to elute the DNA.
      5. Assess 1 μl of material using the Agilent High Sensitivity D1000 ScreenTape system (Appendix III).
    2. PCR addition of indices (2 h)
      Note: Carefully choose your index to allow for multiplexing using the Illumina pooling guidelines.
      1. Perform PCR as described below.


      2. Ampure XP SPRI bead cleanup:
        1. Transfer reaction to a lowBind DNA 1.5 ml microtube.
        2. Add 90 μl beads, pipette up and down 10 times, allow to bind at RT for 5 min.
        3. Place on the DynaMag, discard liquid when clear.
        4. Add 500 μl of fresh 80% ethanol without removing from the DynaMag. Avoid disturbing beads by running the ethanol down the front of the tube. Allow to sit on the rack for 30 sec at RT, then remove ethanol.
        5. Remove ethanol and add another 500 μl of fresh 80% ethanol, allow to sit for 30 sec at RT and remove all the ethanol.
        6. Spin down in a microfuge and remove residual ethanol with a P10 pipette.
        7. Air dry at room temperature on the DynaMag until matt in appearance.
          Note: Be careful not to over dry the beads as this will result in increased DNA losses.
        8. Resuspend beads in 31 μl water, mix by pipetting 10 times.
        9. Incubate at RT for 2 min.
        10. Recover 30 μl.
      3. Assess 1 μl of material using the Agilent D1000 ScreenTape system (Appendix III page 4).
      4. Quantify library using Qubit dsDNA BR assay kit.
      SAFE STOPPING POINT: Store at -20 °C.

  3. Enrichment for viewpoints of interest by oligonucleotide capture
    –Based on the Nimblegen SeqCap SR User’s Guide Chapter 5-7
    Note: The blocking oligonucleotides must be complementary to the adapters in your library. If performing LI-Capture-C, you can use the commercially available blocking oligonucleotides from Nimblegen. If you are following the Tag-Capture-C protocol, you can contact IDT and design blocking oligonucleotides using their xGen Custom Blocking Oligos service. We used a universal blocker for both the i5 adapter and i7 adapters, so we only needed 2 blocking oligonucleotides for all possible index combinations.
    1. Hybridization preparation (1 day)
      Note: Ensure capture oligonucleotides for different experiments are kept apart. The oligonucleotides are hugely in excess and tiny amounts of contamination can lead to spurious results. We would recommend that you do not order oligonucleotides that should not be mixed from the manufacturer at the same time. Avoid having buffers and blocking oligonucleotides out at the same time as the capture oligonucleotides. Oligonucleotides that are going to be pooled for a single experiment, can be ordered together. Consider ordering large designs in as a pooled set (at equimolar concentrations).
      Biotinylated capture oligonucleotides
      1. Reconstitute individual or pools of oligonucleotides to a stock concentration so that each oligo is stored at 2.9 μM (10 nmol in 3.46 μl of PCR grade water)–or any high concentration.
      2. Generate pools of oligonucleotides by mixing in exact 1:1 stoichiometric ratio.
      Nimblegen blocking oligonucleotides (LI-Capture-C)
      Resuspend Nimblegen oligonucleotides according to the Nimblegen protocol:
      1. Spin down tubes in a minifuge.
      2. Add 120 μl PCR grade water to the HE Universal Oligo tube (1 mM); vortex and spin briefly.
      3. Add 10 μl PCR grade water to the HE Index Oligo tube (1 mM); vortex and spin briefly.
      4. Add 480 μl PCR grade water to the Post-LM-PCR Oligos; vortex and spin briefly.
      Custom blocking oligonucleotides (Tag-Capture-C)
      Resuspend the blocking oligonucleotides according to manufacturer’s instructions.
      Multiplexing of samples
      Mix up to 1 μg of each differentially indexed sample at exactly 1:1 ratios by mass to generate a multiplexed library.
    2. Hybridization (3 days)
      1. Heat vacuum centrifuge to 50 °C.
      2. Prepare hybridization reaction for the number of pooled libraries–up to 6 libraries are captured in a single tube. For more libraries, a master mix may be made in one tube and divided into multiple tubes.
        5 μg COT DNA (5 μl of stock)/library
        1 nmol TS-HE Universal Oligo (1 μl of 1 mM stock aliquot)/library
        1 nmol of TS-HE Index Oligos (1 μl of 1 mM stock for each index)
        up to 1 μg of each uniquely indexed 3C library
        Note: For fewer than 100,000 cells you may not recover > 1 μg after indexing. Use as much as possible.
      3. Vacuum centrifuge at 50 °C with tube lids open (rather than pierced) until sample is completely dry. Avoid drying for a long time after the liquid is gone.
      4. For each library in the capture reaction add:
        7.5 μl 2x hybridization buffer (vial 5)
        3 μl hybridization component A (vial 6)
      5. Carefully reconstitute the DNA by pipetting and vortexing followed by briefly spinning down.
      6. Replace buffers and blocking oligonucleotides in freezer prior to proceeding.
      7. Preheat the Eppendorf ThermoMixer to 95 °C.
      8. Preheat a PCR block to 47 °C, the lid should be heated to 57 °C.
      9. Heat 4.5 μl per library of pooled biotinylated oligonucleotide probes to 47 °C in a PCR tube.
        Note: The capture will be at 47 °C for ~72 h. A high-quality PCR tube is required to avoid sample loss through evaporation.
      10. Denature the 3C library and blocking oligonucleotides by heating to 95 °C for 10 min.
      11. After 10 min, quickly spin the denatured library and add the entire volume to the pooled biotinylated oligonucleotides without removing from the thermocycler.
      12. Mix and spin briefly before replacing in the 47 °C PCR machine for 64-72 h.
    3. Washing and recovery of captured material (1 day)
      Notes:
      1. Heat reagents on an Eppendorf ThermoMixer as it is more reliable than a water bath and allows the samples to be shaken.
      2. For capture of multiplexed libraries, scale the beads and wash buffers accordingly to the number of libraries.
      3. The Stringent Wash buffer, Wash Buffer I and Bead Wash Buffer are quite voluminous and may need to be made in multiple tubes. Split Wash Buffer I 1:2 as one third is heated.

      1. Dilute the wash buffers as per table below which allows a slight excess of each.
        1. Place the Stringent Wash Buffer at 47 °C on the ThermoMixer.
        2. Place 100 μl Wash Buffer I per captured library with an additional 20 μl at 47 °C on the ThermoMixer.

          Note: The streptavidin beads will likely stick to the interior of the tube. This is particularly a problem with some makes of low bind tubes. Use tubes with minimal adhesion. We use Safeseal tubes (Sorenson) for this step.
      2. Prepare the streptavidin beads (M270):
        1. Allow the beads to heat to RT for 30 min prior to use.
        2. Aliquot 100 μl per captured library into a single 1.5 ml Eppendorf.
        3. Place on the DynaMag and remove liquid once clear.
        4. Add 200 μl of 1x Bead Wash Buffer per captured library and vortex to resuspend the beads, spin briefly.
        5. Replace on the DynaMag for 5 min and remove liquid once clear.
        6. Repeat wash steps (C3b.iii-C3b.v) for a total of two washes.
        7. Resuspend the beads in their original volume (i.e., 100 μl per captured library) with Bead Wash Buffer (1x) and aliquot into an appropriate number of tubes 1.5 ml Eppendorf tubes.
        8. Place on the DynaMag, remove and discard the liquid only when ready to add the capture sample from the thermocycler. Do not allow beads to dry out.
      3. Binding of biotinylated oligonucleotides:
        1. Transfer the hybridization reactions to the streptavidin beads and mix thoroughly by pipetting 10 times.
        2. Spin briefly if necessary to pool all of the sample in the bottom of the tube.
        3. Place on the ThermoMixer at 47 °C, 600 rpm for 45 min. Mix the beads with a pipette every 15 min to avoid beads settling at the bottom of the tube.
        Note: Steps C3d.i-C3d.vii should be done quickly to maintain the temperature at 47 °C.
      4. Washing the streptavidin beads and bound DNA:
        1. Add 100 μl of heated Wash Buffer I (47 °C) per captured library to the beads and bound DNA.
        2. Mix by vortexing.
        3. Place on the DynaMag, carefully discard all the supernatant when clear.
        4. Add 200 μl Stringent Wash buffer per captured library heated to 47 °C and mix.
        5. Incubate for 5 min at 47 °C.
        6. Place the tubes on the DynaMag, carefully discard the supernatant when clear.
        7. Repeat stringent wash (C3d.iv-C3d.vi) for a total of 2 washes.
        8. Add 200 μl Wash Buffer I (1x) at RT per captured library and mix by vortexing for 2 min. Spin briefly to ensure no sample is lost in the lid.
        9. Place the tubes on the DynaMag, carefully discard the supernatant when clear.
        10. Add 200 μl Wash Buffer II (1x) per captured library and mix by vortexing for 1 min. Spin briefly to ensure no sample is lost in the lid.
        11. Place the tubes on the DynaMag, carefully discard the supernatant when clear.
        12. Add 200 μl Wash Buffer III (1x) and mix by vortexing for 30 sec. Spin briefly to ensure no sample is lost in the lid.
        13. Place the tubes on the DynaMag, carefully discard the supernatant when clear.
        14. Remove from the DynaMag and resuspend beads in 40 μl PCR grade water per capture library. Do not discard beads–DNA is not eluted but amplified off the beads.
        15. Store at -20 °C or proceed to PCR amplification.
          SAFE STOPPING POINT: Store at -20 °C.
    4. PCR amplification of captured material (1 day)
      1. Amplify the captured fragments in two separate reactions per captured library in a total of 14 cycles.


        Note: Only use 20 μl of DNA/Bead–using 40 μl has the potential to inhibit the reaction. Two separate PCR reactions can be performed or 20 μl can be stored at -20 °C.
      2. Ampure XP SPRI bead cleanup:
        1. Transfer reactions to lowBind DNA 1.5 ml microtubes. Combine up to six reactions.
        2. Add 90 μl beads per reaction, pipette 10 times to mix, allow to bind at RT for 5 min.
        3. Place on the DynaMag, discard liquid when clear.
        4. Add 500 μl of fresh 80% ethanol without removing from the DynaMag. Avoid disturbing beads by running the ethanol down the front of the tube. Allow to sit on the rack for 30 sec at RT, then remove ethanol.
        5. Remove ethanol and add another 500 μl of fresh 80% ethanol, allow to sit for 30 sec at RT and remove all the ethanol.
        6. Spin down in a microfuge and remove residual ethanol with a P10 pipette.
        7. Air dry at room temperature on the DynaMag until matt in appearance.
          Note: Be careful not to over dry the beads as this will result in increased DNA losses.
        8. Resuspend beads in 51 μl water, mix by pipetting 10 times.
        9. Incubate at RT for 2 min.
        10. Recover 50 μl.
      3. Assess 1 μl of material using the Agilent D1000 ScreenTape system to confirm same profile as input material.
      4. Quantify with Qubit BR Kit.
      SAFE STOPPING POINT: Store at -20 °C.
    5. Double capture (3 days)
      Note: Capture efficiency is improved by 100-1,000 fold by performing a second capture step. This increases the proportion of captured reads from 1% to 50%. This means only ~1 million reads are needed per viewpoint per sample and thus reduces sequencing requirements. Therefore, double capture is standard practice in our lab.
      1. Pool amplified material of the parallel PCR reactions.
      2. Prepare the second capture hybridization reaction using all captured material (up to 2 μg) in a single reaction. Combine:
        1. 5 μg COT DNA (5 μl of stock)
        2. 1 nmol TS-HE Universal Oligo (1 μl of 1 mM stock aliquot)
        3. 1 nmol of TS-HE Index Oligos (1 μl of 1 mM stock in total; so if you have 4 samples, use 0.25 μl of each index for a total of 1 μl; consider making a dilution if you have many samples)
        4. Up to 2 μg of amplified captured material
      3. Perform hybridization, washes, and amplification as described with a 24-h hybridization at 47 °C and treating the material as a single library.
      SAFE STOPPING POINT: Store at -20 °C.
    6. Library quantification and sequencing (1 day)
      1. Use qPCR with the KAPA Library Quantification Kit to calculate the concentration of adaptor containing fragments.
        Make 1:10,000 and 1:20,000 dilutions of the captured material for quantification.
      2. Sequence using Illumina paired-end sequencing with either 150 bp or 75 bp reads (300-cycle and 150-cycle kits).

Data analysis

The data analysis of low-input Capture-C experiments has been described (Oudelaar et al., 2017). Scripts are available in the GitHub repository (https://github.com/oudelaar/CaptureC/).

Recipes

  1. Fresh lysis buffer, cool to 4 °C–for 10 samples (5 ml):
    10 mM Tris pH 8 (50 μl 1 M)
    10 mM NaCl (10 μl of 5 M)
    0.2% Igepal CA-630 (100 μl of 10%)
    1x cOmplete Protease Inhibitor Cocktail (200 μl of 25x)
    4.64 ml PCR grade water

Acknowledgments

The low-input Capture-C protocols are optimized adaptations from the Next-Generation Capture-C method (Davies et al., 2016). We thank the Wellcome Trust (Wellcome Trust Genomic Medicine and Statistics PhD Programme, reference 105281/Z/14/Z; Wellcome Trust Strategic Award, reference 106130/Z/14/Z) and the Medical Research Council (MRC Core Funding and Centenary Award reference 4050189188) for funding our work. The authors declare that there is no conflict of interest.

References

  1. 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.
  2. Davies, J. O., Oudelaar, A. M., Higgs, D. R. and Hughes, J. R. (2017). How best to identify chromosomal interactions: a comparison of approaches. Nat Meth 14: 125-134.
  3. Davies, J. O., Telenius, J. M., McGowan, S. J., Roberts, N. A., Taylor, S., Higgs, D. R. and Hughes, J. R. (2016). Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat Methods 13(1): 74-80.
  4. Dekker, J., Rippe, K., Dekker, M. and Kleckner, N. (2002). Capturing chromosome conformation. Science 295(5558): 1306-1311.
  5. Oudelaar, A. M., Davies, J. O. J., Downes, D. J., Higgs, D. R. and Hughes, J. R. (2017). Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C. Nucleic Acids Res 22(45): e184.

简介

染色体构象捕获(3C)技术对于理解基因表达的组织特异性调节是至关重要的,但是目前的方法通常需要大量的细胞。 该协议描述了两种新的低输入Capture-C方法,根据用于分析的分辨率,可以从10,000-20,000个细胞生成高质量的3C相互作用谱。

【背景】3C技术在调查调控元件之间的核组织和结构相互作用与基因活性之间起关键作用(Dekker等人,2002)。 由于这些相互作用是高度组织特异性的,3C定义的纯化细胞群进行3C实验是至关重要的。

3C技术的一个主要局限性是所需要的大量细胞:目前的方法使用了10万到10万个细胞(Davies等人,2017)。 这些数字中不包含许多原发性组织和稀有细胞群。 因此,我们开发了两种新的低输入Capture-C方法,可以从最大分辨率的〜20,000个细胞(单独的DpnII片段)和使用基于开窗分析的约10,000个细胞产生高质量的相互作用谱(Oudelaar等人 。,2017)。

关键字:核结构, 染色质构型, 染色体构象捕获(3C), Capture-C, 低样品量, 少量细胞

材料和试剂

  1. 3C图书馆准备
    1. LowBind DNA 1.5毫升微管
    2. 锁相凝胶(PLG)灯管(VWR,Quantabio,目录号:733-2477)
    3. 基因组DNA ScreenTape(Agilent Technologies,目录号:5067-5366)
    4. Tapestation加载技巧(安捷伦科技,产品目录号:5067-5153)
    5. 1M甘氨酸(Sigma-Aldrich,目录号:G7126)
    6. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010031)
    7. 成长媒体
    8. 37%甲醛(Sigma-Aldrich,目录号:252549)
    9. 无水乙醇&gt; 99.8%(VWR,产品目录号:20821.330,或同等产品)
    10. 干冰或液氮
    11. PCR级别的水(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9932)
    12. SDS(20%v / v水溶液)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9820)。
    13. Triton X-100(20%v / v水溶液)(Sigma-Aldrich,目录号:T8787)
    14. (50,000U / ml)(New England Biolabs,目录号:R0543M)
    15. T4 DNA连接酶(30U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EL0013)。
    16. 蛋白酶K(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0491)
    17. RNase(无DNA酶)(Roche Diagnostics,目录号:11119915001)
    18. 苯酚 - 氯仿 - 异戊醇(PCI)25:24:1(Sigma-Aldrich,目录号:77617)
    19. 3M NaOAc(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9740)
    20. GlycoBlue(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9515)
    21. 基因组DNA试剂(Agilent Technologies,目录号:5067-5365)
    22. 不含UNG的TaqMan Universal PCR Master Mix II(Thermo Fisher Scientific,Applied Biosystems TM,产品目录号:4440040)
    23. KAPA Sybr Fast Universal(卡帕生物系统,目录号:KK4602)
    24. 新鲜的裂解缓冲液(见食谱)
      1. Tris pH8(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9855G)
      2. 氯化钠(NaCl)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9760G)
      3. Igepal CA-630(100μl,10%)(Sigma-Aldrich,目录号:I8896)
      4. 完整蛋白酶抑制剂混合物(在2ml PCR级水中1片,25x;储存于-20℃)(Sigma-Aldrich,Roche Diagnostics,目录号:11873580001)
      5. PCR级水
    25. qPCR引物(附录I

  2. 通过连接或标记添加Illumina测序适配器来生成Capture-C文库 的 LI-捕获-C
    1. Covaris microTUBE AFA纤维预分割卡入式6 x 16 mm(Covaris,目录号:520045)
    2. LowBind DNA 1.5毫升微管
    3. PCR管
    4. D1000 ScreenTape(Agilent Technologies,目录号:5067-5582)
    5. TapeStation加载技巧(安捷伦科技,产品目录号:5067-5153)
    6. 用于Illumina的NEBNext Ultra DNA文库制备试剂盒(New England Biolabs,目录号:E7370S / L)
    7. Agencourt安普XP SPRI珠(贝克曼库尔特,目录号:A63881)
    8. 用于Illumina Primer set 1(New England Biolabs,目录号:E7335S / L)的NEBNext Multiplex Oligos
    9. 用于Illumina引物组2的NEBNext多重寡核苷酸(New England Biolabs,目录号:E7500S / L)
    10. Herculase II融合聚合酶试剂盒(Agilent Technologies,目录号:600677)
    11. PCR级别的水(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9932)
    12. 无水乙醇&gt; 99.8%(VWR,产品目录号:20821.330,或同等产品)
    13. D1000试剂(Agilent Technologies,目录号:5067-5583)
    14. Qubit dsDNA BR分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32850)

    的 标记捕获-C
    1. LowBind DNA 1.5毫升微管
    2. PCR管
    3. 高灵敏度D1000 ScreenTape(安捷伦科技,产品目录号:5067-5584)
    4. D1000 ScreenTape(Agilent Technologies,目录号:5067-5582)
    5. TapeStation加载技巧(安捷伦科技,产品目录号:5067-5153)
    6. Nextera DNA样品制备试剂盒(Illumina,目录号:FC-121-1030 / 1031)
    7. Zymo DNA Clean&amp; Concentrator-5试剂盒(Zymo Research,产品目录号:D4013)
    8. 带有dNTP的KAPA HiFi PCR试剂盒(KK2102)(Roche Diagnostics,目录号:07958846001)
    9. 定制设计的指数引物
    10. Agencourt安普XP SPRI珠(贝克曼库尔特,目录号:A63881)
    11. PCR级别的水(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9932)
    12. 无水乙醇&gt; 99.8%(VWR,产品目录号:20821.330,或同等产品)
    13. 高灵敏度D1000试剂(Agilent Technologies,目录号:5067-5585)
    14. D1000试剂(Agilent Technologies,目录号:5067-5583)
    15. Qubit dsDNA BR分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32850)

  3. 通过寡核苷酸捕获富集感兴趣的观点
    1. Safeseal微量离心管(Sorenson BioScience,目录号:39640T)
    2. D1000 ScreenTape(Agilent Technologies,目录号:5067-5582)
    3. Tapestation加载技巧(安捷伦科技,产品目录号:5067-5153)
    4. Nimblegen SeqCap EZ杂交和洗涤试剂盒(Roche Diagnostics,目录号:05634261001)
    5. Nimblegen SeqCap EZ附件试剂盒v2(Roche Diagnostics,产品目录号:07145594001)
    6. 1μg/μl相关物种的COT DNA(小鼠)(Thermo Fisher Scientific,Invitrogen TM,目录号:18440016)
    7. M-270抗生蛋白链菌素Dynabeads(Thermo Fisher Scientific,Invitrogen TM,目录号:65305)
    8. Agencourt安普XP SPRI珠(贝克曼库尔特,目录号:A63881)
    9. PCR级别的水(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9932)
    10. Qubit dsDNA BR分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32850)
    11. Qubit dsDNA HS分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32851)
    12. D1000试剂(Agilent Technologies,目录号:5067-5583)
    13. KAPA Library Quantification Complete Kit(Universal)(Kapa Biosystems,目录号:KK4824)
    14. LI-Capture-C
      Nimblegen SeqCap EZ HE-oligo试剂盒A(Roche Diagnostics,目录号:06777287001)
      Nimblegen SeqCap EZ HE-oligo试剂盒B(Roche Diagnostics,目录号:06777317001)
    15. Tag-Capture-C
      xGen Custom Blocking Oligos(P5 / i5&amp; P7 / i7 Nextera拦截器)

设备

  1. 离心机
  2. 孵化器(任何)
  3. Eppendorf Thermomixer C(或相当的振荡培养箱)(Eppendorf,型号:ThermoMixer C,目录号:5382000015)
  4. P10,P20,P200和P1000移液器(任何)
  5. 热循环仪(任何)
  6. NanoDrop
  7. Agilent 2200 TapeStation(Agilent Technologies,型号:Agilent 2200 TapeStation系统)
    注意:安捷伦TapeStation上的评估也可以在安捷伦的生物分析仪上进行。
  8. 超声波仪(Covaris,型号:S220聚焦超声波仪)
  9. DynaMag-2(Thermo Fisher Scientific,目录号:12321D)
  10. 真空浓缩机(SciQuip,CHRIST系列)

程序

概述
注意:程序概述(图形改编自Davies等人,2017)。


的步骤

  1. 3C图书馆准备
    1. 固定(2小时)
      注:以下描述的固定程序已经针对红细胞进行了优化。取决于使用的细胞类型,可能需要调整一些离心步骤以确保适当的造粒。
      1. 预冷离心机至4°C。冷藏甘氨酸,PBS和裂解缓冲液(见食谱)。
      2. 从组织中收集细胞并在1ml生长培养基中制备10,000到10 6个细胞的单细胞悬浮液。如果分选细胞,分入100μl培养基,然后加入培养基,总体积为1 ml。
      3. 加入54微升37%甲醛(终浓度2%),充分混匀,室温摇动或摇动培养箱孵育10分钟。
      4. 通过加入150μl1M冷甘氨酸(终体积的1/8即1.2ml)淬灭。

      5. 离心15分钟/ 500×g / 4℃
      6. 小心取出上清液,不要搅动沉淀。可以留下〜5%。
      7. 通过轻轻悬浮在1ml冷PBS中洗涤沉淀。

      8. 离心15分钟/ 500×g / 4℃
      9. 小心取出上清液,不要搅动沉淀。可以留下〜5%。
      10. 重悬沉淀在500μl冷裂解缓冲液。

      11. 在冰上孵育20分钟
      12. 用乙醇和干冰或液氮快速冷冻。快速冷冻辅助消化,以便细胞可以在此解冻再消化,或在-80°C长期保存。
        安全停止点:储存在-80°C。
    2. 消化(1天)
      1. 将ThermoMixer预热至37°C。
      2. 准备消化混合(为多个图书馆做一个主混合):
        PCR grade water
        152.5 μl
        10x DpnII Buffer
        20 μl
        20% SDS
        2.5 μl

      3. 每个反应的等分10,000到10 6个甲醛固定细胞
      4. 离心10分钟/ 15,000×g / 4℃。

      5. 。小心地取出所有的裂解缓冲液,使用P20去除残余的缓冲液而不影响沉淀。
      6. 重悬于175μl的消化混合物。

      7. 使用ThermoMixer,在37°C水平(1,400 rpm)振荡1 h
      8. 加入16μl的20%Triton X-100(终浓度为1.7%),并在37℃振荡1小时。
      9. 加入第一等份的3微升的emp Dpn II酶,并在37℃摇动至少两个小时。
      10. 在一天结束时加入第二份3μl的emp Dpn II酶并摇动过夜。

      11. 第二天早上再加入3微升的emp Dpn II酶,再摇动至少三个小时。
    3. 结扎(1天)
      1. 将消化物置于65°C封闭20分钟以加热限制酶。
      2. 在冰上冷却消化,冷却ThermoMixer到16°C。
      3. 使连接混合(为多个库建立一个主混合):
         PCR grade water
        125 μl
        10x ligation buffer
        36 μl
      4. 添加161微升连接混合消化。
      5. 添加4微升T4连接酶,并在300转/ 16°C摇晃〜22小时。
    4. 去交联(1天)
      加入2微升蛋白酶K(20毫克/毫升),并在65°C孵育过夜。
    5. DNA提取(1天)
      1. 向连接反应中加入4μlRNase,并在37℃孵育30分钟。
      2. 加入350μl苯酚 - 氯仿 - 异戊醇并彻底涡旋。
      3. 转移到2毫升的PLG灯管,旋转5分钟/ 15,000 em x em / em / RT。
      4. 将上层,〜350微升,转移到一个新的管。
      5. 加入35μl3 M NaOAc和2μlGlycoBlue,倒置混匀。
      6. 加入960μl100%乙醇,倒置混合。

      7. 在-20°C过夜冷冻 安全停止点:DNA应至少沉淀一晚;这可以延长到几天,而不会影响恢复。

      8. 在孵化过程中预冷离心机至4°C
      9. 离心45分钟/ 21,000×g / 4℃,小心丢弃液体。
      10. 用1毫升70%的冰冷乙醇洗沉淀。
      11. 自旋10分钟/ 21,000×g / 4℃。
      12. 去除乙醇,重复乙醇洗(步骤A5j-A5k)。
      13. 去除乙醇,在微型离心机中旋转,用吸管去除残留的乙醇。
        在室温下干燥并重悬沉淀。处理LI-Capture-C时,重悬于124μl水中。对于Tag-Capture-C,水量取决于输入量,因为几个标记反应是并行设置的(下面解释)。每15,000个细胞使用20μl的水,向上取整(例如,30,000个细胞:40μl水; 50,000个细胞:80μl水)。
        安全停止点:-20°C储存。
    6. 量化和QC(3小时)
      注意:由于连接缓冲液中残留的DTT,NanoDrop或类似的分光光度计对于定量3C文库是不可靠的。
      1. 使用Agilent Genomic ScreenTape系统评估1μl3C文库以确定文库的大小分布(附录I )。
      2. 对于LI-Capture-C,取3μl3C文库和27μl水稀释1/10;为标记捕获C - 取1微升的3C文库和29微升的水,使1/30稀释。
        进行qPCR消化效率,使用稀释。基因组DNA可以用作未消化的对照(附录I )。消化效率应该> 75%。

  2. 通过连接或标记添加Illumina测序适配器来生成Capture-C文库 LI-Capture-C
    - 基于用于Illumina(E7370)的NEBNext Ultra DNA文库制备试剂盒的方案 注意:
    1. 在这个步骤中,通过最大化输入DNA和减少反应和清理过程中的DNA损失来保持文库的复杂性是非常重要的。当有超过1微克的DNA时,进行平行准备。这些可以在清理和最后清理。
    2. NEB方案通常建议用比方案中下一步所需的水少数多的水从珠子中去除DNA。人们可以避免这样做,以最大限度地减少损失,但这意味着要非常小心珠清理,因为珠或乙醇的污染可以抑制下面的反应。
    3. 我们使用Covaris S220聚焦超声仪进行超声处理。如果使用不同的模型超声波仪,使用高分子量的gDNA来优化超声波的大小约为200 bp的模式分布( 附录II )。
    4. 您添加到文库中的Illumina指标需要与捕获步骤中所需的Nimblegen HE阻断寡核苷酸(试剂盒不匹配)互补; )附录二 同时购买引物组1和引物组2,消除了更昂贵的Nimblegen阻断试剂盒的浪费。
    5. 不需要选择大小,因为接头二聚体不会被捕获。所有Ampure XP珠子清理都是用1.8倍体积的珠子进行的。

    1. 超声处理(1小时)
      1. 取120微升的3C文库(高达1微克),并转移到Covaris微管。避免泡沫。
      2. 将DNA剪切至200bp,使用以下设置:
        工作周期:10%
        强度:5
        每次爆发周期:200
        时间:360秒
        设置模式:扫频
      3. 安培XP SPRI珠清理:
        1. 从Covaris微管转移到低结合DNA 1.5毫升微管。
        2. 加入220μl珠子,上下移液10次,使其在RT下结合5分钟。
        3. 放在DynaMag上,清除时丢弃液体。
        4. 加入500μl新鲜的80%乙醇,不从DynaMag中移出。通过在管子前面运行乙醇避免干扰珠子。
          允许在室温下搁置30秒,然后除去乙醇。
        5. 除去乙醇,再加入500μl新鲜的80%乙醇,放置30秒钟,除去所有乙醇。
        6. 在微离心机上旋转,用P10移液管除去残留的乙醇。

        7. 在DynaMag的室温下风干,直到表面无光泽。
          注意:小心不要过度干燥珠子,因为这会导致DNA损失增加。
        8. 将珠子重悬于56μl水中以洗脱DNA,通过移液10次混合。
        9. 在室温下孵育2分钟。
        10. 恢复55.5微升的修复反应。
      4. 可选:使用Agilent D1000(常规或高灵敏度,取决于您的输入)评估1μl超声处理材料ScreenTape系统(附录II )。
      安全停止点:-20°C储存。
    2. 末端修复和适配器结扎(2小时)
      1. 将55.5μlDNA(5ng-1μg),6.5μl10x末端修复反应缓冲液(绿色)和3μl末端制备酶混合物(绿色)组合。通过移液混合并短暂旋转。
      2. 在热循环仪中孵育:
        20°C 30分钟
        65°C 30分钟
      3. 加入15μlBlunt / TA连接酶主混合物(红色),2.5μlNEBNext衔接物*(红色),1μl连接增强物质(红色)。通过移液混合并短暂旋转。
        注意:如果DNA输入是&lt; 100 ng,在10 mM Tris-HCl或含10 mM NaCl的10 mM Tris-HCl中稀释10倍的Illumina NEBNext Adaptor(15μM提供),终浓度为1.5μM,立即使用。 br />

      4. 在20°C下,在热循环仪中孵育15分钟
      5. 加入3μlUSER酶(红色)。通过移液混合。

      6. 在37°C的热循环仪中孵育15分钟
      7. 安培XP SPRI珠清理:
        1. 转移反应到一个低结合DNA 1.5毫升微管。
        2. 加入160微升珠子,上下吸移10次,使其在室温下结合5分钟。
        3. 放在DynaMag上,清除时丢弃液体。
        4. 加入500μl新鲜的80%乙醇,不从DynaMag中移出。通过在管子前面运行乙醇避免干扰珠子。
          允许在室温下搁置30秒,然后除去乙醇。
        5. 除去乙醇,再加入500μl新鲜的80%乙醇,放置30秒钟,除去所有乙醇。
        6. 在微型离心机中旋转,用P10移液管移除残留的乙醇。

        7. 在DynaMag的室温下风干,直到表面无光泽。
          注意:小心不要过度干燥珠子,因为这会导致DNA损失增加。
        8. 在29μl水中重悬微珠,移液10次。
        9. 在室温下孵育2分钟。
        10. 将28.5μl洗脱的DNA转移到PCR管中。
    3. 指标的PCR添加(2小时)
      注意:请仔细选择您的索引,以便使用Illumina池化指南进行复用。
      1. 执行如下所述的PCR。


      2. 安培XP SPRI珠清理:
        1. 转移反应到一个低结合DNA 1.5毫升微管。
        2. 加入90微升珠子,上下吸移10次,允许在室温下结合5分钟。
        3. 放在DynaMag上,清除时丢弃液体。
        4. 加入500μl新鲜的80%乙醇,不从DynaMag中移出。通过在管子前面运行乙醇避免干扰珠子。
          允许在室温下搁置30秒,然后除去乙醇。
        5. 除去乙醇,再加入500μl新鲜的80%乙醇,放置30秒钟,除去所有乙醇。
        6. 在微型离心机中旋转,用P10移液管移除残留的乙醇。

        7. 在DynaMag的室温下风干,直到表面无光泽。
          注意:小心不要过度干燥珠子,因为这会导致DNA损失增加。

        8. 在31μl水中重悬微珠,移液10次
        9. 在室温下孵育2分钟。
        10. 恢复30μL。
      3. 使用Agilent D1000 ScreenTape系统评估1μl材料(附录II第3页)。
      4. 使用Qubit dsDNA BR分析试剂盒量化文库。
      安全停止点:-20°C储存。

    Tag-Capture-C
    注意:
    1. 在这个步骤中,通过最大化输入DNA和减少反应和清理过程中的DNA损失来保持文库的复杂性是非常重要的。标签化反应的最大输入量是50ng。当输入较高时,并行运行几个独立的反应;这些可以在清理之后合并。
    2. 我们使用自定义设计的索引引物,可以在附录中找到它们的序列。我们将这些订单从IDT订购为TruGrade Primers。
    3. 不需要选择大小,因为接头二聚体不会被捕获。所有Ampure XP珠子清理都是用1.8倍体积的珠子进行的。

    1. 标签(1天)
      1. 使用PCR管中Nextera DNA样品制备试剂盒中的试剂准备标记反应:
        &LT; 50微升3C文库在20微升水中
        25微升TD缓冲液
        5μlTD Enzyme
      2. 通过移液混合,然后快速旋转。

      3. 在55°C孵育12-24小时,以确保所有的材料被贴上标签
      4. Zymo旋转柱清理:
        1. 标签管和添加180微升Zymo DNA结合缓冲液。
        2. 加入50μl标记的DNA。轻轻地上下移动10次混合。
        3. 将样品混合物转移到收集管中的Zymo离心柱上。
        4. 在15,000×g g下离心30秒。丢弃流通。
        5. 添加200微升Zymo DNA洗涤缓冲液的列。
          15,000×g离心30秒。
        6. 重复步骤B1d.v(标记 - 捕获 - C部分)并另外在15,000×g克上离心60秒。
        7. 转移到一个1.5毫升管的列,并添加34.5微升的水柱基质。在室温下孵育2分钟。

        8. 15,000×g离心1分钟以洗脱DNA。
      5. 使用安捷伦高灵敏度D1000 ScreenTape系统评估1μl材料(附录三)。
    2. 指标的PCR添加(2小时)
      注意:请仔细选择您的索引,以便使用Illumina池化指南进行复用。
      1. 执行如下所述的PCR。
        “”

      2. 安培XP SPRI珠清理:
        1. 转移反应到一个低结合DNA 1.5毫升微管。
        2. 加入90微升珠子,上下吸移10次,允许在室温下结合5分钟。
        3. 放在DynaMag上,清除时丢弃液体。
        4. 加入500μl新鲜的80%乙醇,不从DynaMag中移出。通过在管子前面运行乙醇避免干扰珠子。
          允许在室温下搁置30秒,然后除去乙醇。
        5. 除去乙醇,再加入500μl新鲜的80%乙醇,放置30秒钟,除去所有乙醇。
        6. 在微型离心机中旋转,用P10移液管移除残留的乙醇。

        7. 在DynaMag的室温下风干,直到表面无光泽。
          注意:小心不要过度干燥珠子,因为这会导致DNA损失增加。

        8. 在31μl水中重悬微珠,移液10次
        9. 在室温下孵育2分钟。
        10. 恢复30μL。
      3. 使用Agilent D1000 ScreenTape系统评估1μl材料(附录III第4页)。
      4. 使用Qubit dsDNA BR分析试剂盒量化文库。
      安全停止点:-20°C储存。

  3. 通过寡核苷酸捕获来丰富感兴趣的观点
    - 基于Nimblegen SeqCap SR用户指南第5-7章
    注意:阻断寡核苷酸必须与文库中的接头互补。如果执行LI-Capture-C,您可以使用Nimblegen的市售阻断寡核苷酸。如果您遵循Tag-Capture-C方案,您可以联系IDT并使用xGen Custom Blocking Oligos服务设计阻断寡核苷酸。我们对i5适配器和i7适配器都使用了通用阻断剂,所以我们只需要2个阻断寡核苷酸来进行所有可能的指数组合。
    1. 杂交准备(1天)
      注意:确保不同实验的捕获寡核苷酸保持分开。寡核苷酸极其过量,微小的污染可能导致虚假的结果。我们建议您不要同时订购不应该由制造商混合的寡核苷酸。避免与捕获寡核苷酸同时将缓冲液和寡核苷酸封闭。将被汇集进行单个实验的寡核苷酸可以一起排序。考虑订购大型设计作为一个集合(等摩尔浓度)。
      生物素化的捕获寡核苷酸
      1. 将寡核苷酸个体或寡核苷酸重新配成原液浓度,以便每种寡核苷酸保存在2.9μM(10 nmol,在3.46μlPCR级别的水中)或任何高浓度。
      2. 以精确的1:1化学计量比混合产生寡核苷酸池。
      Nimblegen阻断寡核苷酸(LI-Capture-C)
      根据Nimblegen方案重新悬浮Nimblegen寡核苷酸:

      1. 在微型离心机中旋下管
      2. 向HE Universal Oligo试管(1 mM)中加入120μlPCR级别的水;漩涡和短暂旋转。
      3. 向HE指数Oligo管(1mM)中加入10μlPCR级水;漩涡和短暂旋转。
      4. 将480μlPCR级别的水添加到Post-LM-PCR Oligos;涡旋并短暂旋转。
      定制阻断寡核苷酸(Tag-Capture-C)
      根据制造商的说明重新封闭寡核苷酸。
      样品复用
      以1:1的比例按质量比混合1μg差异标记的样品,以产生多重文库。
    2. 杂交(3天)
      1. 加热真空离心机至50°C。
      2. 准备杂交反应的数量的汇集库 - 多达6库收集在一个单一的管。对于更多的图书馆,主混合可能在一个管,并分成多个管。
        5微克COT DNA(5微升股票)/图书馆
        1 nmol TS-HE Universal Oligo(1μl1 mM储液等分试样)/ library
        1 nmol TS-HE指数寡核苷酸(每个指数1μl的1毫升原液)
        每个独特索引的3C图书馆最多1μg
        注意:对于少于100,000个单元,您可能无法恢复&gt;索引后1微克。尽可能多地使用。
      3. 在50℃真空离心机中,管盖打开(而不是穿孔),直到样品完全干燥。
        在液体消失后避免长时间的干燥
      4. 对于捕获反应中的每个文库,添加:
        7.5μl2x杂交缓冲液(小瓶5)
        3μl杂交成分A(小瓶6)
      5. 通过吸取和涡旋小心地重组DNA,然后短暂地旋转。

      6. 将Eppendorf ThermoMixer预热至95°C。
      7. 将PCR块预热至47°C,盖子应加热至57°C。

      8. 每个文库加入4.5μl生物素标记的寡核苷酸探针,加热至47°C 注意:捕获将在47°C〜72小时。需要使用高质量的PCR管来避免蒸发造成的样品损失。
      9. 变性3C文库,并通过加热到95°C 10分钟阻断寡核苷酸。
      10. 10分钟后,快速旋转变性的文库,并将整个体积加入合并的生物素化寡核苷酸,而不从热循环仪中移出。

      11. 在47℃PCR仪上更换64-72小时之前,轻轻混合并旋转
    3. 捕获物质的清洗和回收(1天)
      注意:
      1. 在Eppendorf ThermoMixer上加热试剂,因为它比水浴更可靠,可以摇动样品。
      2. 为了捕获多路复用的文库,根据库的数量来缩放珠粒并洗涤缓冲液。
      3. 严格的洗涤缓冲液,洗涤缓冲液I和珠洗涤缓冲液相当丰满,可能需要在多个管中进行。将三分之一洗涤缓冲液I 1:2三分之一加热。

      1. 按照下面的表格稀释洗涤缓冲液,每个缓冲液略微过量。

        1. 将ThermoMixer的Stringent洗涤缓冲液置于47°C

        2. 每个捕获的文库放置100μl洗涤缓冲液I,在ThermoMixer上47°C再加20μl。

          注:链霉抗生物素蛋白珠可能会粘附在管内部。这对于一些低绑定管的制造来说尤其是个问题。用最小的粘附力使用管子。我们使用Safeseal管(Sorenson)进行这一步。
      2. 准备链霉亲和素珠(M270):

        1. 在使用前,让珠子加热至RT 30分钟

        2. 每个捕获的文库分装100微升到一个1.5毫升的Eppendorf
        3. 放在DynaMag上,一旦清除就移除液体。
        4. 每捕获一个文库,加入200μl1x Bead洗涤缓冲液,涡旋重悬珠子,短暂旋转。

        5. 在DynaMag上取代5分钟,然后清除液体。
        6. 重复洗涤步骤(C3b.iii-C3b.v)共两次洗涤。
        7. 用珠粒洗涤缓冲液(1x)重悬其原始体积的珠子(即每个捕获的文库100μl),并将其等分至适当数量的1.5ml Eppendorf管中。
        8. 置于DynaMag上,只有在准备好从热循环仪中加入捕获样品时,才能取出并丢弃液体。 不要让珠子变干。
      3. 生物素化寡核苷酸的结合:
        1. 将杂交反应转移到链霉亲和素珠上,用移液器充分混合10次。

        2. 如果需要,可以短暂旋转,将所有样品收集在试管底部
        3. 放在ThermoMixer上,在47℃,600转45分钟。每15分钟用移液管混合珠子,以避免珠子沉降在管子的底部。
        注意:步骤C3d.i-C3d.vii应该尽快完成,以保持温度在47℃。
      4. 洗涤抗生蛋白链菌素珠和结合的DNA:

        1. 每个捕获的文库加入100μl加热的洗涤缓冲液I(47℃)至珠子并结合DNA
        2. 通过涡旋混合。
        3. 放在DynaMag上,清除时要小心丢弃所有的上清液。

        4. 每个捕获的文库加入200μlStringent Wash缓冲液,加热至47°C并混合

        5. 在47°C孵育5分钟
        6. 将管放在DynaMag上,清除时小心丢弃上清液。
        7. 重复严格洗涤(C3d.iv-C3d.vi)共2次。
        8. 在每个捕获的文库中加入200μl洗涤缓冲液I(1x),涡旋混合2分钟。短暂旋转以确保没有样品丢在盖子里。
        9. 将管放在DynaMag上,清除时小心丢弃上清液。
        10. 每个捕获的文库添加200μL洗涤缓冲液II(1x),涡旋混合1分钟。短暂旋转以确保没有样品丢在盖子里。
        11. 将管放在DynaMag上,清除时小心丢弃上清液。
        12. 加入200μl洗涤缓冲液III(1x)并涡旋混合30秒。短暂旋转以确保没有样品丢在盖子里。
        13. 将管放在DynaMag上,清除时小心丢弃上清液。
        14. 从DynaMag中取出并用40μlPCR级别的水重悬微珠,每个捕获文库。不要丢弃珠子DNA不会被洗脱,而是从珠子上被放大。
        15. 保存在-20°C或进行PCR扩增。
          安全停止点:-20°C储存。
    4. 捕获物质的PCR扩增(1天)

      1. 每个捕获的文库在两个独立的反应中扩增捕获的片段,共14个循环。


        注意:只使用20微升的DNA /珠 - 使用40微升有抑制反应的潜力。可以进行两个独立的PCR反应,或者20μl可以在-20℃下保存。
      2. 安培XP SPRI珠清理:
        1. 将反应转移至低结合DNA 1.5ml微管。结合多达六个反应。
        2. 每次反应添加90微升珠,移液器10次混合,允许在室温下结合5分钟。
        3. 放在DynaMag上,清除时丢弃液体。
        4. 加入500μl新鲜的80%乙醇,不从DynaMag中移出。通过在管子前面运行乙醇避免干扰珠子。
          允许在室温下搁置30秒,然后除去乙醇。
        5. 除去乙醇,再加入500μl新鲜的80%乙醇,放置30秒钟,除去所有乙醇。
        6. 在微型离心机中旋转,用P10移液管移除残留的乙醇。

        7. 在DynaMag的室温下风干,直到表面无光泽。
          注意:小心不要过度干燥珠子,因为这会导致DNA损失增加。

        8. 在51μl水中重悬微珠,移液10次
        9. 在室温下孵育2分钟。
        10. 恢复50μL。
      3. 使用Agilent D1000 ScreenTape系统评估1μl材料,以确认与输入材料相同的性能。
      4. 用Qubit BR Kit量化。
      安全停止点:-20°C储存。
    5. 双重捕捉(3天)
      注意:通过执行第二个捕获步骤,捕获效率提高100-1,000倍。这将捕获的阅读比例从1%增加到50%。这意味着每个样本每个观察点只需要约100万个读取,因此降低了测序要求。因此,双重捕捉是我们实验室的标准操作。
      1. 并行PCR反应池的扩增材料。
      2. 准备第二次捕获杂交反应使用所有捕获的材料(高达2微克)在一个单一的反应。结合:
        1. 5微克COT DNA(5微升股票)
        2. 1 nmol TS-HE Universal Oligo(1μl1 mM储备液)
        3. 1 nmol TS-HE指数寡核苷酸(1μl总共1 mM);所以如果您有4个样品,请使用0.25μl的各个指标,总共1μl;如果您有多个样品,请考虑稀释)。 />
        4. 多达2微克的扩增捕获材料
      3. 按照所述进行杂交,洗涤和扩增,在47℃下进行24小时杂交,并将材料作为单一文库进行处理。
      安全停止点:-20°C储存。
    6. 文库量化和测序(1天)
      1. 使用qPCR与KAPA文库量化试剂盒来计算包含片段的接头浓度。
        对捕获的物质进行1:10,000和1:20,000稀释以进行定量。
      2. 使用Illumina双末端测序的序列(150个碱基对或75个碱基对的读取)(300个循环和150个循环的试剂盒)。

数据分析

已经描述了低输入俘获-C实验的数据分析(Oudelaar等人,2017)。脚本可以在GitHub仓库中找到( https://github.com/oudelaar/CaptureC/ )。

食谱

  1. 新鲜的裂解缓冲液,冷却到4°C - 10个样品(5毫升):
    10 mM Tris pH 8(50μl1 M)
    10mM NaCl(10μl5M)
    0.2%Igepal CA-630(100μl,10%)
    1x cOmplete蛋白酶抑制剂鸡尾酒(200μl25x)
    4.64毫升PCR级水

致谢

低输入Capture-C协议是来自新一代Capture-C方法(Davies等人,2016)的优化改编。我们感谢Wellcome Trust(Wellcome Trust Genomic Medicine and Statistics PhD Program,参考文献105281 / Z / 14 / Z; Wellcome Trust战略奖,参考文献106130 / Z / 14 / Z)和医学研究理事会(MRC Core Funding and Centenary Award参考文献4050189188)资助我们的工作。作者声明不存在利益冲突。

参考

  1. Buenrostro,J.D。,Giresi,P.G.,Zaba,L.C.,Chang,H.Y。和Greenleaf,W.J。(2013)。 天然染色质的转换,用于开放染色质,DNA结合蛋白和核小体位置的快速和敏感的表观基因组谱分析。 Nat方法 10(12):1213-1218。
  2. Davies,J.O.,Oudelaar,A.M.,Higgs,D.R。和Hughes,J.R。(2017)。 如何最好地鉴定染色体相互作用:方法比较 Nat Meth 14:125-134。
  3. Davies,J.O.,Telenius,J.M.,McGowan,S.J.,Roberts,N.A.,Taylor,S.,Higgs,D.R。和Hughes,J.R。(2016)。 染色体构象的多重分析,灵敏度大大提高 Nat Methods,
    13(1):74-80
  4. Dekker,J.,Rippe,K.,Dekker,M。和Kleckner,N。(2002)。 捕获染色体构象 科学 295(5558): 1306-1311。
  5. Oudelaar,A.M。,Davies,J.O.J。,Downes,D.J。,Higgs,D.R。和Hughes,J.R。(2017)。 使用低密度脂蛋白对少量细胞进行稳定的染色体相互作用检测,输入Capture-C。 Nucleic Acids Res 22(45):e184。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Oudelaar, A., Downes, D. J., Davies, J. O. and Hughes, J. R. (2017). Low-input Capture-C: A Chromosome Conformation Capture Assay to Analyze Chromatin Architecture in Small Numbers of Cells. Bio-protocol 7(23): e2645. DOI: 10.21769/BioProtoc.2645.
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