ChIPseq protocol

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Courtesy of Andrea Wills (UW), Rakhi Gupta, Edward Chuong and Julie Baker (Stanford). See ref for more details

Sample Preparation

Materials

  • Methanol-free formaldehyde (Sigma F8775)
  • 0.125M Glycine
  • 1X PBS
  • Magnetic particle concentrator (Invitrogen 123.21D )
  • Dynabeads protein A (Invitrogen 10001D)
  • Dynabeads protein G (Invitrogen 10003D)
  • Antibody (diluted according to manufacturer instructions)
  • PBS+5% BSA: 250mg BSA (Sigma A9647-50G) in 50ml PBS (Gibco 10010-023). Store at 4° and discard after one week.
  • RIPA buffer (4°C, 1.25 ml per set of 50 embryos): 50 mM Tris-HCl, pH 7.4, 1% Igepal CA-630 (NP-40) (Sigma I3021), 0.25% Na-Deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5 mM DTT, 5 mM Na-Butyrate
  • Complete, Mini Protease Inhibitor Cocktail Tablets (Roche 11836153001), 1 tablet per 10mL RIPA buffer.

Equipments

  • Rotator or Nutator, at 4°C
  • Sonicator
  • Refrigerated centrifuge

Day 1

  1. Culture embryos to desired stage.
  2. Fix embryos for 1 hour in 1% formaldehyde/PBS
  3. Wash 5 minutes in 0.125 M Glycine
  4. Wash 3 times, quickly, in PBS. Divide embryos into batches of up to 100 X. laevis or 250 X. tropicalis.
  5. Remove all PBS and freeze up to 6 months at -80°C, or proceed directly to Day 2 steps.
  6. Prepare Dynabeads with antibody (see section 3)

Day 2

  1. Thaw crosslinked embryos on ice, 10-15 minutes.
  2. Add 600μl cold RIPA+Protease Inhibitor to each sample.
  3. Break embryos by pipetting with a P1000 tip or by gentle disruption with a plastic pestle, until embryos are broken into small fragments and the solution is gray in color. The embryos need not be completely homogenized yet.
  4. Centrifuge at 14,000 g for 10 minutes using a refrigerated centrifuge.
  5. Decant supernatant, and wipe the walls of the tube with a kimwipe to remove traces of yolk.
  6. Add 100μl cold RIPA, and homogenize thoroughly, taking care to avoid bubbles, until no visible fragments of embryo remain and homogenate is a uniform gray.
  7. Add 550μl cold RIPA.
  8. Chill embryo samples on wet ice for 20-30 minutes, until ready to sonicate. Gently resuspend each sample by pipetting immediately before sonication.
  9. Sonicate using empirically determined conditions. We recommend trying 2, 5, or 10 cycles at 30%, 50% or 100% intensity to start.
  10. Centrifuge at 14,000 g for 10 minutes using a refrigerated centrifuge.
  11. Transfer 60 μL of the resulting lysate to new tubes for input controls.
  12. Transfer supernatant to pre-clear beads, and then to antibody-conjugated beads (see section 3).

Immunoprecipitation

Materials

  • Low salt buffer: 0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 150mM NaCl (Store at 4 ℃)
  • High salt buffer: 0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 500mM NaCl (Store at 4 ℃)
  • LiCl salt buffer: 0.25M LiCl (L-8895 Sigma), 1% IGEPAL CA630 (Sigma I-3021), 1% deoxicholate acid, 1mM EDTA, 10mM Tris-HCL pH8.0 (Store at 4 ℃)
  • TE buffer: 10mM Tris-HCl, 1mM EDTA, pH 8.0 (Store at 4 ℃)
  • TES buffer: 50mM Tris-HCl pH8.0, 10mM EDTA, 1% SDS (store at room temperature)
  • 5M NaCl
  • Phenol/Chloroform/Isoamyl Alcohol (Thermo Fisher BP1752)
  • Chloroform (Thermo Fisher BP1145)
  • Sodium Acetate buffer solution (Sigma S-7899)
  • Glycogen (Fermentas cat# R0561 Fermentas)
  • Qiagen PCR Purification Kit (Qiagen 28004)
  • RNase A (Roche 10109169001)
  • 70% and 100% Ethanol

Day 1 (the day before homogenization and sonication)

  1. Resuspend protein G or protein A Dynabeads by vortexing. Use 50 μL dynabeads for each experiment, including mock pull-down, if performing. All DynaBeads can be aliquoted in one volume and washed together.
  2. Place tubes in bead separator, wait until beads have migrated against magnet and sample is clear.
  3. Wash beads twice with 1 mL of PBS+5% BSA, vortexing beads well and then returning to magnet for each wash.
  4. Resuspend beads in PBS+5% BSA, using 200 μL for each experiment. Vortex beads well and aliquot into individual tubes for each experiment. Bring volume up to 1 ml (+800 μL) with PBS+5% BSA.
  5. Add primary antibody according to manufacturer-recommended dilutions.
  6. Place tubes on a nutator or rotator at 4°C overnight.

Day 2 (same day as sonication)

  1. Prepare 20 μL of washed Dynabeads per sample as above, resuspending in 1 mL PBS+5% BSA. These are the “pre-clear” beads.
  2. Place against magnet to pellet pre-clear beads, discard supernatant, and replace with the supernatant of sonicated, centrifuged embryo lysate.
  3. Incubate pre-clear beads and lysate with rotation at 4°C for one hour.
  4. Use magnet to pellet ANTIBODY-conjugated beads. Remove and discard supernatant. Use magnet to pellet PRE-CLEAR beads, and transfer supernatant (lysate) from pre-clear beads onto antibody- conjugated beads. Discard pre-clear beads.
  5. Incubate antibody- conjugated beads plus lysate 4°C overnight, with rotation.

Day 3

  1. Use magnet to pellet antibody-conjugated beads, discard supernatant. Perform washes at 4°C, using 1 ml of solution for each wash, and using the magnet to pellet beads between each wash:
    • Low salt solution (2 washes, 5 minutes each)
    • High salt solution (2 washes, 5 minutes each)
    • LiCl buffer (2 washes, 5 minutes each)
    • TE (2 washes, 5 minutes each)
  2. Remove all TE, and replace with 200μl TES. Return to room temperature.
  3. Vortex beads very thoroughly, let settle, and vortex again.
  4. Incubate beads at 65 degrees for 15 minutes, vortexing every 5 minutes.
  5. Vortex thoroughly once more, pellet beads using magnet, and transfer supernatant carefully to new, labeled microfuge tube.
  6. Remove input samples from freezer and thaw.
  7. Add 16μl 5M NaCl to input samples and immunoprecipitated samples. Cap lids tightly and incubate at least 5 hours or overnight at 65°C. (Optional: for higher-quality DNA, include 7μl proteinase K/Glycogen solution).

Day 4

  1. Add 1 volume (approx. 200μL) Phenol/Chloroform to samples. Vortex until milky, and centrifuge at 12,000 g for 5-7 minutes.
  2. Transfer supernatant to new, clean, labeled microfuge tube.
  3. Repeat extraction with 1 volume (200μl) Chloroform; vortex, spin and transfer supernatant as above.
  4. Add 1/10 vol (20μl) 3M Sodium acetate, 2.5 vol (500μl) 100% Ethanol, and 1μl glycogen. Precipitate at least 5 hours or overnight at -20°C.
  5. Centrifuge at full speed for 15 minutes, taking care to note the orientation of the tubes. Small pellets should be visible in input samples, but may not be visible in IP samples.
  6. Carefully remove supernatant and wash pellets with 500μl 70% ethanol.
  7. Centrifuge at full speed for 1 minute, taking care to note the orientation of the tubes and position of pellets. Carefully remove all traces of supernatant.
  8. Resuspend in 15μl of nuclease-free water and quantify yield as described below. Alternatively, if the resulting DNA will be used for qPCR, perform the following additional steps:
    1. Incubate in 100ul RNAse A/TE for 1 hour at 37°C.
    2. Purify using a Qiagen Minelute reaction purification kit, according to kit instructions, using 15μl as the final elution volume
    3. Quantify yield using nanodrop. If yield is low (<50ng/μl), use a high sensitivity method such as Qbit to accurately quantitate yield.

Library Preparation

Materials

All inclusive kits:

  1. Genomic DNA prep kit from Illumina (requires Qiagen Minelute reaction cleanup reagents and columns as well)
  2. TruSEQ kit from Illumina Piecemeal:

• T4 ligase and buffer. Invitrogen 15224-017 • T4 DNA Polymerase. NEB M0203L • 10mM dNTPs. Invitrogen 18427-013 • Klenow DNA Polymerase • T4 PNK • Minelute reaction cleanup kit. Qiagen 28204 • dATP. Invitrogen 10216-018 • Klenow Fragment (3’-5’ Exo). NEB M0212L • HPLC-purified Adapters (consult Illumina for sequences) • E. coli DNA ligase. Invitrogen 18052-019 • 2% eGel. Invitrogen G6610-02 –or- • NuSieve GTG agarose. Lonza 50080 • 50bp ladder. Invitrogen 10416-014 • Phusion high-fidelity 2X master mix polymerase kit. NEB M0532S • HPLC-purified primers (consult Illumina for sequences)

End Repair

  1. Aliquot enough ChIP DNA to reach 1μg (minimum) or 5μg (ideal). Bring up DNA samples to 30ul with nuclease-free water.
  2. Repair ends by preparing the following reaction mix, using PCR tubes:
    • ChIP DNA 30μl
    • Nuclease free water 45μl
    • T4 ligase buffer 10μl
    • dNTPs 4μl
    • T4 DNA polymerase 5μl
    • Klenow DNA Polymerase 1μl
    • T4 PNK 5μl
    • Total 70μl
  3. Incubate at 20°C for 30 minutes
  4. Clean up the reaction using Qiagen Minelute reaction clean up kit, as follows (or follow manufacturer instructions):
  5. To the 70μl reaction mix, add 300μl buffer ERC, mix.
  6. Pipet into Minelute column inside collection tube.
  7. Centrifuge at full speed one minute.
  8. Discard flow-through.
  9. Add 750ul Buffer PE to the top of column, centrifuge 1 minute.
  10. Discard flow through, and centrifuge again 1 minute to remove traces of ethanol.
  11. Transfer Minelute column to new, clean, labeled microfuge tube.
  12. Add 32μl buffer nuclease-free water to the middle of each column, let sit one minute.
  13. Centrifuge 1 minute at full speed.


Adenylate ends

  1. Transfer samples to PCR tubes.
  2. To each sample (32μl), add:
    • Klenow Buffer 5μl
    • dATP 10μl
    • Klenow exonuclease (3’ to 5’ exo) 3μl
    • Total 50μl
  3. Incubate at 37°C 30’.
  4. Clean up reaction using Minelute column as above, eluting in 18μl final volume.
  5. Transfer samples to PCR tubes.

Adapter Ligation

  1. Ligate adapters by preparing the following reaction mix:
    • End-repaired DNA 18μl
    • DNA ligase buffer 25μl
    • Adapter oligo mix 2μl
    • DNA ligase 5μl
    • Total: 50μl
  2. React at 20°C for 15 minutes.
  3. Gel-purify samples using either a 2% e-Gel or 3% Nuseive Agarose gel (see above).

eGel purification

  1. Load samples into upper wells, leaving a space between each sample to avoid contamination. Choose one lane to run a 50bp ladder.
  2. Load 20ul water into any empty top wells, and all bottom wells.
  3. Select “2%” mode. Run eGel for 13 minutes.
  4. Use UV illumination to check position of ladder. DNA in samples should be visible as a smear. Add 10μl water to each bottom well.
  5. Run gel slowly, checking frequently, until bottom wells are positioned between the 200 and 250 bp band. Isolate these bands from each sample. If the gel runs too long, change mode to “reverse e-Gel” and run in reverse

until the desired band comes into the bottom wells.

  1. If desired, add 20μl additional water to bottom wells, and run gel until 300bp band is positioned in bottom well. Isolate these bands from samples.

Agarose gel purification

  1. Prepare 3% gel using NuSeive GTG agarose and TAE
  2. Combine samples with 10X loading dye. Also prepare a 1:10 dilution of 50bp ladder with loading dye.
  3. Load samples, leaving an empty well between each sample to avoid contamination.
  4. Run gel according to usual methods. Check by UV visualization; the DNA should be visible as a smear in wells containing samples.
  5. Use a clean scalpel blade to isolate the 200-250bp region.
  6. Purify DNA from gel fragment using Minelute gel purification kit, following manufacturer instructions, and eluting in 20μl final volume.

Amplification

  1. Amplify the library by preparing the following PCR reaction:
    • Gel-purified DNA 4μl
    • Phusion DNA polymerase
    • 2X Master Mix 25μl
    • Primer 1.1 1μl
    • Primer 2.1 1μl
    • Water 19μl
    • Total 50μl
  2. Perform the following PCR reaction, using 15 cycles if the initial amount of ChIP DNA was 1μg, or 12 cycles if the initial amount of DNA was 2μg or more.
    • 30 seconds at 98°C
    • [10 seconds at 98°C
    • 30 seconds at 65°C
    • 30 seconds at 72°C] X 12-15 cycles
    • 5 minutes at 72°C
  3. Purify the resulting PCR products using a Minelute PCR cleanup kit, following manufacturer instructions, eluting in 20μl final volume.
  4. Check DNA concentration using a high-sensitivity method, such as Qbit or Bioanalyzer. If concentrations are too low (below 3ng/μl), repeat PCR and combine and concentrate reactions.