ChIP

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This protocol is in development.~ cmc 15:03, 21 August 2006 (EDT)

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

11% Formaldehyde Solution

per 50 ml: (Final concentration)

  • 14.9 ml of 37% Formaldehyde (11%)
  • 1 ml of 5M NaCl (0.1M)
  • 100 µl of 0.5 M EDTA, pH 8 (1 mM)
  • 50 µl of 0.5 M EGTA, pH 8 (0.5 mM)
  • 2.5 ml of 1M Hepes, pH 8 (50 mM)

2.5 M glycine (187.5 g/L)

  • Dissolve glycine in water with constant stirring
  • Don’t adjust pH

BSA/PBS Solution

per 100 mL:

  • 10 mL of 10x PBS
  • 500 mg of BSA
  • 90 mL of H20

(good for at least a week)

Lysis Buffer 1 (LB1)

per 100 ml: (final concentration)

  • 5 ml of 1M Hepes-KOH, pH 7.5 (50 mM)
  • 2.8 ml of 5M NaCl (140 mM)
  • 0.2 ml of 0.5M EDTA (1 mM)
  • 10 ml of glycerol (10%)
  • 5 ml of 10% NP-40 (0.5%)
  • 0.25 ml of Triton X-100 (0.25%)

Lysis Buffer 2 (LB2)

per 100 ml: (final concentration)

  • 4 ml of 5M NaCl (200 mM)
  • 0.2 ml of 0.5M EDTA (1 mM)
  • 0.1 ml of 0.5M EGTA (0.5 mM)
  • 1.0 ml of 1M Tris pH 7.5 (10 mM)

Lysis Buffer 3 (LB3)

per 100 ml: (final concentration)

  • 0.2 ml of 0.5M EDTA (1 mM)
  • 0.1 ml of 0.5M EGTA (0.5 mM)
  • 1.0 ml of 1M Tris-HCl, pH7.5 (10 mM)
  • 2 mL of 5M NaCl (100 mM)
  • 1 mL of 10% Na-Deoxycholate (0.1%)
  • 500 mg of N-lauroyl sarcosine (0.5%)

Wash buffer (RIPA buffer)

per 100ml: (final concentration)

  • 5ml of 1M Hepes, pH 7.6 (50 mM)
  • 200µL of 0.5M EDTA (1 mM)
  • 7 ml of 10% DOC (Na deoxycholate) (0.7%)
  • 10 ml of 10% NP-40 (IPGEL) (1%)
  • 10 ml of 5M LiCl or 2.12g powder (0.5 M)

Elution buffer

  • 50mM Tris pH8
  • 1mM EDTA
  • 1% SDS

Oligos for blunt end ligation

  • oJW102: 5’-GCGGTGACCCGGGAGATCTGAATTC
  • oJW103: 5’-GAATTCAGATC
  • Final buffer/concentrations for annealing:
    • 50 mM Tris pH 7.5
    • 50 mM NaCl
    • 17.5 µM each oligo
  • After mixing, boil 5 minutes, and anneal slowly from a 100°C heat block to 25°C, then cool to 4°C overnight, aliquot, and freeze.



Procedure

I. Cell cross-linking

  • Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each ChIP reaction.
  • Adherent cells:
    1. Add 1/10 volume of fresh 11% formaldehyde solution to plates.
    2. Swirl plates briefly and let them sit at RT for 10 min.
    3. Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde.
    4. Rinse cells twice with 5 ml 1X PBS. Harvest cells using silicon scraper.
    5. Spin cells at 4k for 10’ at 4°C.
    6. Transfer cells to 15ml conical tubes and spin 4k 10’ at 4°C.
    7. Flash freeze cells in liquid nitrogen and store pellets at –80 °C.
  • Suspension cells:
    1. Add 1/10 volume of fresh 11% formaldehyde solution to flasks.
    2. Swirl flasks briefly and let them sit at RT for 20 min.
    3. Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde.
    4. Spin cells at 2500rpm for 10’ at 4°C.
    5. Rinse cells with 100ml 1x PBS and spin cells at 2500rpm for 10’ at 4°C.
    6. Rinse cells with 10ml 1x PBS and transfer to 15ml conical tubes and spin 4k 10’ at 4°C.
    7. Flash freeze cells in liquid nitrogen and store pellets at –80 °C.

II. Preblock and binding of antibody to magnetic beads

  • Keep beads on ice for all steps
  1. wash 100 µL Dynal magnetic beads (per reaction) in 1 ml fresh BSA/PBS
  2. collect the beads with magnet wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet.
  3. Add 6-10 µg of Ab + 250µL of PBS/BSA.
  4. incubate 4 hr to O.N. on a rotating platform at 4°C.
  5. wash beads 3 times in 1.5 ml PBS/BSA.
  6. Resuspend in 100µL PBS/BSA.


III. Cell Sonication

  • Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml H2O for 25x solution. Use 400uL per 10mL of buffer. Store in aliquots at -20°C.
  1. Resuspend each tube of cells in 5-10 ml of Lysis buffer I. Rock at 4°C for 10’. Then spin at 2500rpm in a table top centrifuge, 3 min at 4°C.
  2. Resuspend each tube of cells in 5-10 ml of buffer 2. Rock gently at 4°C for 5 min. Pellet nuclei in tabletop centrifuge by spinning at 2500 rpm, 3 min at 4°C.
  3. Resuspend pellet in each tube in 3 ml buffer 3 and put into 15mL conical tube cut off at the 6.5mL mark.
  4. Sonicate the suspension using the automatic sonicator.
    • Appropriate sonication time needs to be determined for each cell type. For example, HepG2 cells: 6 cycles of 30 seconds with 1 minute intervals at power 7.0 (~40 watts).
  5. Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes. Spin out debris 14K, 4°C, 10 min.
  6. If you are using one tube of cells for more than one ChIP then you need to add more LB3 and 10% Triton X-100 so that the volume ends up being 3 mL of cells in LB3 with 300uL of Triton per ChIP.
  7. Save 50 µL (or at least 1/50 vol) of cell lysate from each sample as input control. Store at -20°C.


IV. Chromatin Immunoprecipitation

  1. Add 100 µL Ab prebound Dynal magnetic beads from step III.
  2. Rock 4°C 6 hrs to O/N.

V. Washing, eluting, and reverse cross-linking

  • Work in the cold room until step 6.
  1. Transfer IP reactions to centrifuge tubes
  2. Use the magnetic stand to precipitate the beads.
  3. Wash 4-8 times with 1 mL wash buffer (Ripa buffer)
  4. Wash once with 1 ml TE-plus-50 mM NaCl or 1 mL TBS.
  5. Spin 3k for 2-3 min and aspirate any residual TE/TBS.
  6. Add 200 µl of elution buffer.
  7. Elute DNA-protein complexes from beads at 65°C for 10-15 min with brief vortexing every 2 min.
  8. Spin down beads 14k for 1 min.
  9. Remove all 200 µl of supernatant.
  10. Reverse x-link O/N 65°C. Min 6 hours
  11. Thaw input from step III(6), add 3 vol of elution buffer and reverse x-link O/N 65°C.


VI. RNase, Proteinase K

  1. Add 1 vol of TE to IP and input (wce) fraction.
  2. Add RNase A so final is 0.2µg/µL (~5 µL/250 µL rxn). Incubate 37∞C 1-2hr.
  3. Add proteinase K so final is 0.2µg/µL (~2.5 µL/250 µL rxn). Incubate 55∞C 1-2hr.
  4. Extract once w/ 1 vol of phenol.
  5. Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol).
  6. extract once w/ 1 vol of chl:IA.
    • Alternatively: extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes
  7. add 20 µg (1 µL) of glycogen.
  8. Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn).
  9. Add 2X volume of EtOH. Precipitate DNA with a 10 min 14K spin, and wash pellet with 500 µL 75% EtOH.
  10. Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8.
  11. Normalize the input/wce fraction to 100 ng/µL using the Nanodrop.

VII. T4 DNA Polymerase Fill-in

  1. To 60 µL of IP sample, and 200 ng (=2 µL) of the normalized input (wce) diluted to 60 µL, add:
    • 10 µL of 10X T4 DNA pol buffer
    • 0.5 µL of BSA (NEB 10mg/ml)
    • 0.4 µL of 25mM dNTP mix
    • 0.2 µL of T4 DNA pol (NEB 3U/µL)
    • 28.9 µL of H2O
    • final volume =100uL
    • Incubate 12∞C 20 min in water bath.
    • Add 12 µl 3 M NaOAc and 1.0 µL glycogen.
    • Extract 1x with 120 µL phenol:chl:IA.
    • Precipitate with 300 µL EtOH.
    • Spin and wash with 500 µL 75% EtOH.
    • Dry pellet and resuspend in 25 µL H2O.

VIII. Blunt-end ligation

  1. Make at 4°C, 25 µL ligase mix per reaction:
    • 7.8 µL of H2O
    • 10 µL of 5X ligase buffer (Gibco)
    • 6.7 µL of annealed linkers (thaw at 4°C)
    • 0.5 µL of T4 DNA ligase
  2. add mix to 25 µL of sample.
  3. incubate 12∞C O/N, cover with foil.
  4. Next day add 6µL of 3M NaOAc and 150 µL EtOH.
  5. Spin and wash with 500 µL 75% EtOH.
  6. Dry and resuspend pellet in 40 µL PCR reaction mix below.

IX. Ligation mediated PCR (LM-PCR)

  • for PCR: use pellets from blunt ligation.
  1. Make PCR mix per rxn:
    • 35 µL of H2O
    • 4 µL of 10X Thermopol buffer
    • 0.5 µL of 25 mM dNTP mix
    • 0.5 µL of 100 µM oligo oJW102
  2. Dissolve pellet in mix above and start PCR program CHIPCHIP:
    • 55 ∞C, 4’; 72∞C, 3’; 95∞C, 2’; 95∞C, 30’’; 60∞C, 30’’; 72∞C, 1’; goto step 4 14 times; 72∞C, 5’; 4∞C, hold.
  3. During step 1 (55 ∞C, 4’) add TAQ mix:
    • 8 µL of H2O
    • 1 µL of 10X ThermoPol buffer
    • 0.5 µL of TAQ (5U/µL)(Gibco)
  4. Dilute PCR product in 450uL of H2O.
  5. Take 10uL of diluted product and PCR again using PCR mix per rxn:
    • 25 µL of H2O
    • 4 µL of 10X Thermopol buffer
    • 0.5 µL of 25 mM dNTP mix
    • 0.5 µL of 100 µM oligo oJW102
  6. Start PCR program:
    • 55 ∞C, 4’; 72∞C, 3’; 95∞C, 2’; 95∞C, 30’’; 60∞C, 30’’; 72∞C, 1’; goto step 4 24 times; 72∞C, 5’; 4∞C, hold.
  7. During step 1 (55 ∞C _ 4’) add TAQ mix:
    • 8 µL of H2O
    • 1 µL of 10X ThermoPol buffer
    • 0.5 µL of TAQ (5U/µL)(Gibco)
  8. Clean up DNA using Qiagen kit or precipitation by adding 25µL of 7.5M NH4OAc and 300µL EtOH. Spin and wash pellet with 500 µL 75% EtOH. Redissolve in 50 µL H2O.
  9. Normalize [DNA] to 100 ng/µL.

Checkpoints

  1. Sonication Fragment Size Verification
    • After sonication, the spread of size fragments can be checked by running 1 µL of input/wce control DNA on a 1% agarose gel. Spread should extend no higher than 2 kb.
  2. Product Size and Amount Verification
    • Remove 2 µL of PCR product and run it out on 2% agarose gel. Product sizes should be around 250-350 bp. Amount of IP PCR product should be roughly equal to input PCR product. Check the concentration using the Nanodrop, expecting ~50-100 ng/µL.

X. Cy3-Cy5 Random Primer labeling

  1. From the normalized IP and wce PCR DNA above, 1 µg of DNA will be fluorescently labeled (=10µL of LM-PCR product).
  2. Add 24 µL water to above.
  3. Add 26.8 µL of 2.5 X random primer solution (Invitrogen Bioprime labeling kit).
  4. Boil 5 min in heatblock.
  5. Place tubes in icewater. Incubate 5 min.
  6. Add 8 µL 10X low T dNTP mix (1.2 mM dATP, dCTP, dGTP each and 0.6mM dTTP).
  7. Add 1 µL of cy5-dUTP to IP tube and 1 µL cy3-dUTP to input tube.
  8. Add 1 µL of high concentration Klenow (40U/µL, Bioprime kit).
  9. Incubate 20 ∞C 6hrs- O/T (keep samples in dark as much as possible from here on).
    • Alternative: incubate 37 oC for 3 hrs.

To Be Continued ~ cmc 18:18, 21 August 2006 (EDT)


Notes

  1. This protocol will likely fail the first time you try it. Your LM-PCR product will contain no DNA. Don't loose heart. Try, try again. (Hard to pin down exactly what goes wrong between sonication and LM-PCR, but it seems that the blunting step is very sensitive. Most people get it to work by trying two or three times.) ~ cmc 16:25, 21 August 2006 (EDT)
  2. Gene-specific PCR can be used to validate the results of your ChIP-chip.

References

Contacts

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