HiC protocol

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Courtesy of Ian Quigley @ Salk Institute (Kintner Lab)

Crosslinking & Lysis

Examples of starting material: 2 ~ 2.5 x 107 mammalian cell, X. laevis 100 embryos at stage 10.5

  1. Fixed samples (i.e. Xenopus embryo in formaldehyde).
  2. Dounce cells with 550 µL Lysis buffer.
    • Alternatively, use a pestle with 550 µL Lysis buffer with 15 µL of 10% SDS (final concentration 0.56%)
  3. Spin the chromatin at 5,000 rpm, and wash with 1 mL ice-cold Wash buffer. Twice.
    • Use DAPI to check the nuclei under microscope (optional).
    • For frog, remove fats using Kimwipes.

Pellet preparation

  1. Re-suspend the chromatin in 250 µL of Wash buffer.
  2. Add 95 µL of 2 % SDS, mix and incubate at 65 ℃ for 10 minutes. Place tubes back on ice immediately after incubation.
  3. Dilute sample with 650 µL of 1x NEB2, and incubate on ice for 5 min.
    • (Optional) Let cool to RT, add 105 µL of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB, dissolve 6.78 mg in 500 µL water), mix, and rock at RT for 45 min.
  4. Quench the SDS by adding 150 µL Triton X-100, mix carefully, and incubate on ice for 5 min. Then incubate at 37°C for an additional 10 min.
  5. Digest DNA with 35 µL of 25 U/µL MboI (NEB), together with 85 µL 10x NEB2, 30 µl 1 M DTT, and 200 µL water. Mix and incubate using a rotor at 37°C overnight.
    • Alternatively, human cells use 400U of HindIII.
  6. (Optional) Perform dialysis twice against 5 l TE pH 8.0 (0.5 l per sample) for 3 h, then 1 h at 4°C, respectively in 20 kD Slide-A-Lyzer cassettes.
  7. (Optional) Collected dialyzed samples into 2 ml tubes and kept in fridge.

Blunt-end ligation with biotin-dCTP

  1. Fill in the ristriction fragment overhangs with biotin by adding following reagents.
    • 10 mM dATP 1.5 µL
    • 10 mM dTTP 1.5 µL
    • 10 mM dGTP 1.5 µL
    • 0.4 mM Biotin-14-dCTP 37.5 µL
    • Klenow (5 U/µl) 10 µL
  2. Mix carefully and incubate for 45 min at 37°C.
  3. Place the tubes on ice. Inactivate Klenow by adding 86 µL of 10% SDS. Incubate at 65 °C. for 30 min and place them on ice immediately.
  4. The ligation is performed under extremely dilute condition (in 15 mL conical tube).
    • 745 μL of 10% Triton X-100
    • 745 μL of 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT)
    • 80 μL of 10 mg/ml BSA
    • 80 μL of 100 mM ATP
    • 5.96 mL of water
  5. Add 50 μL of 1 U/μL T4 DNA ligase. Mix by inverting the tubes and incubate at 16°C for 4 hours.

Reverse crosslinking & DNA purification

  1. Add 50 µL of 10 mg/ml proteinase K, and digest proteins at 65°C for overnight.
  2. Add another 50 µL of proteinase K and incubate at 65°C for 2 hours.
  3. Cool the samples to room temperature, and transfer them to a 50 mL conical tube.
  4. Add 10 mL of phenol (pH 8.0) and vortex for 2 minutes.
  5. Spin the tube for 10 min at 3,500 rpm, and transfer the aqueous phase to new 50 mL tube.
  6. Repeat the extraction using phenol (pH 8) : chloroform = 1:1 and precipitate hte DNA using ethanol.
  7. Dissolve DNA pellet in 450 µL 1x TE buffer, and transfer it to a centrifuge (eppendorf) tube).
  1. Remove biotin from unligated ends using the exonuclease activity of T4 DNA polymerase


  1. Extract DNA with 400/200 µl phenol/chloroform/IAA and once with 400 200 µl CHCl3. No protein interphase, assume that proteinase K self-digested.
  2. Bring SN to (200~230 mM NaCl with 20 µl 5 M NaCl, and add 1.5 µl 15 mg/ml Glycoblue.
  3. Precipitate with 1060 µl 100% EtOH (2.5x initial volume, total volume ~1500) for 2 h at -80°C.
  4. Pellet DNA for 20’ at 21000x g (15000 RPM), 4°C, wash twice with 1 ml 80% EtOH for 5’. Air-dry, resuspend pellets in 20 µl each. Did not pool aliquots, to not overload column in next step.
  5. Digest for 30’ at 37°C with 1 µl 2 µg/µl RNase A, clean up DNA on Zymo DNA
  6. Clean&Concentrator column (wash 2x with 200 µl WB) into 20 µl EB, pool aliquots of #4.
  7. Digest the purified DNA sample was treated with 300/20 units of Exo III (NEB) in a total volume of 90/30 μL of NEBuffer1 for 1 hour at 37 C26°C for 1h, 37°C for 30’’The
  8. reaction was stopped by adding 2/0.5 μL of 0.5 M EDTA and 2/0.5 μL of 5 M NaCl, and the enzyme was inactivated by incubation at 70 Cfor 20 minutes.
  9. Shear with 200 bp Covaris protocol, the 90 µl in 104 µl final in snap cap tubes, the 30 µl in 51 µl final in screw top tubes with the requisite Covaris protocols.
  10. Clean up with 155/77.5 µl PEG8000/1.5 M NaCl/ 5/2.5µl Ser-Mag beads.
    • To large samples, add 74.5 µl water, 0.5 µl 10 % Tween 20, 10 µl 10x T4 DNA ligase buffer, 4 µl (0.4 mM) 10 mM dNTP, and end-repair with 5 µl (15 U) T4 DNA polymerase, 1 µl (5 U) Klenow, 5 µl (50 U) T4 PNK for 30’at 20°C.
  11. Clean up DNA with 160/80 µl 20% PEG/1.5M NaCl, dual 80% EtOH washes.
  12. Resuspend in 50 µl EBT, freeze ON.
  1. A-tail by adding 5.9 µl 10x NEB2, 0.12 µl (0.2 mM) 100 mM dATP (7.5x MM: 4.25 µl 10x NEB2, 0.9 µl 100 mM dATP, add 6/5.6 µl each) and incubation with 3 µl/0.3 µl (15 U) exo--Klenow for 3040’ at 37°C.
  2. Capture DNA with 15/5 µl T1 Dynabeads instead of 10 µl C1 Dynabeads, use 0.1 % Triton-X100 in wash buffer (all washes in 500 µl buffer), 0.1% Tween 20 in ligation reaction:
  3. Wash 66 µl beads twice with 1x B&W buffer (2X B&W: 10mM Tris-HCl pH=7.5, 1 mM EDTA, 2 M NaCl), resuspend in 246.4 µl 2x B&W containing 0.2% Tween 20, add 56/18.7 µl of the suspension +0 µl/37.3 µl 2x B&W containing 0.2% Tween 20 to each A-tailing reaction and rotate for 30’ at RT.
  4. Wash once with 1x B&W/0.1% Triton-X100, once with TE.
  5. Resuspend beads in 100/30 µl 1x rapid ligation buffer with 0.1% Tween 20, 0.5 µl undiluted (5 M cells) or 1 µl 1:20 diluted Bioo adapters, 5/1 µl HC ligase, and incubate 20’ at RT.
  6. Stop reaction with 6 µl 0.5 M EDTA, wash twice with 1x B&W, twice with TETET, then resuspend beads in 30 40 µl 0.033% Tween20/LoTE, keep on ice.
  7. Test PCR with 0.66 µl bead suspension for 14/18/22 cycles each:
  8. Make 3.3x MM: 31.35 µl MM + 1.65 µl bead suspension.
  1. Test whether free biotin increases PCR efficiency:
  2. PCR with 0.25 µl bead suspension of sample #5 (sonicated) for 14/17/20 cycles each:
  3. Make 3.3x MM: 15.675 µl MM + 0.825 µl bead suspension.

Pull-down

DNA LoBind tubes are required.

  1. Wash 150 µL resuspended magnetic straptavidin beads (2x) with 400 µL Tween buffer.
  2. Add Buffer to beads.
  3. Transfer the mixture to a new tube.
  4. Rotate the sample for 3 min at room temperature.
  5. Remove the supernatant with holding the magnet.
  6. Resuspend the beads in 300 uL of 2x No Tween Buffer, and combined with 300 uL Hi-C DNA.
  7. Rotate the sample for 15 minutes at room temperature.
  8. Collect the DNA bound streptavidin beads with magnet, and remove the supernatant.
  9. Wash the beads in 400 uL 1x NTB, followed by 100 uL 1x ligation buffer.
  10. Resuspend the beads in 50 uL 1x ligation buffer and transfer the mixture to a new tube.

Library Preparation

  1. 6 pL of Illumina paired-end adapters per uG of HiC DNA available for ligation.
  2. Add 1,200 U T4 DNA ligase, and incubate for 2 hours at room temperature.
  3. Remove non-ligated paired-end

  1. Use 20 µL bead suspension (40x more than test) in 2x 50 µL, run 12 cycles,
  2. Double SpeedBead cleanup with 8.6% PEG final (99.5 µl PCR product (kept 0.5 µL product in in 4.5 µL water for sizing), 2 µl Speedbeads, 76.5 µl 20% PEG/2.5 M NaCl),
  3. first time elute into 50 µl,
  4. second time into 20 µl.
  5. Check size/adapters of samples 6 without, after first and after second cleanup on TapeStation -
  6. if adapter dimers persisted, run on 2% agarose gel without EtBr, stain with SYBRgold, gel-extract 250-550 bp fragments on Minelute column into 20 µl EB.

Buffers

  • Lysis buffer 10 mL
    • 1 M HEPES (pH 8.0) : 100 µL (final 10 mM)
    • 5 M NaCl : 20 µL (final 10 mM)
    • 10% IGEPAL CA-630 : 200 µL (final 0.2 %)
    • 50x Complete Protease Inhibitor Cocktail (Roche) : 200 µL [Add fresh]
    • Water : 9.48 mL
  • Wash buffer 50 mL
    • 1 M Tris/HCl (pH 8.0) : 2.5 mL (final 50 mM)
    • 5 M NaCl : 500 µL (final 50 mM)
    • 0.5 M EDTA : 100 µL (final 1 mM)
    • Water : 46.9 mL

Reagents

  • EZlink Iodoacetyl-PEG2-Biotin Pierce
  • MboI NEB
  • Slide-A-Lyzer 20 kDa G2 Pierce
  • MyOneT1 Life Technologies
  • dGTPαS Axxora
  • Biotin-14-dCTP Life Technologies
  • T4 DNA ligase HC/Enzymatics Enzymatics
  • Exonuclease III NEB
  • Proteinase K (20 mg/ml) Ambion
  • PC Life Technologies
  • Glycoblue Life Technologies
  • Zymo-5 Zymo Research
  • Covaris snap-cap microTube Covaris
  • T4 DNA polymerase/Enzymatics Enzymatics
  • Klenow polymerase/Enzymatics Enzymatics
  • T4 polynucleotide kinase/Enzymatics Enzymatics
  • Exo- Klenow (Enzymatics) Enzymatics
  • Sera-Mag Thermo
  • MyOneT1 Life Technologies
  • T4 DNA ligase HC/Enzymatics Enzymatics
  • PCR master Q5 NEB NEB

See also