ChIP

Overview

Chromatin Immunoprecipitation determines the in vivo chromatin binding sites of a transcription factor or other protein of interest.
Use this protocol in conjunction with ChIP-chip and/or Gene-specific PCR.

Materials

This protocol is in development. Need to add further info about reagents/equipment. ~ cmc 11:43, 22 August 2006 (EDT)

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
  
  • Dissolve the BSA in H2O before adding the PBS.
  • Can make a liter, filter, aliquot by 200 mL, and store at 4°C. Good for months.

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 (or work at 4°C).

1. wash 100 µL of Protein G 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.
   • Don't forget this!!

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

• Check that you have a waterbath or incubator at 65°C.
• 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 washes are sufficient for most antibodies.
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. Transfer all 200 µl of supernatant to a clean 1.5 mL tube.
   • Alternatively: pulse samples to pull beads off the lid, but leave beads in until after the reverse x-link incubation.
9. Reverse x-link at 65°C. Min 6 hours, up to O/N.
10. Thaw input (wce) samples from step III(6), add 3 vol of elution buffer and reverse x-link in parallel with IP samples, 6 hrs - o/n.

VI. RNase, Proteinase K

1. Remove beads from IP samples, if you didn't earlier.
2. Add 1 vol of TE to IP and input (wce) fraction.
3. Add RNase A so final is 0.2µg/µL (~8 µL/400 µL rxn). Incubate 37°C 1-2hr.
4. Add proteinase K so final is 0.2µg/µL (~4 µL/400 µL rxn). Incubate 55°C 1-2hr.
5. Extract once w/ 1 vol of phenol.
6. Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol).
7. extract once w/ 1 vol of chl:IA.
   • Alternatively: extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes
8. add 20 µg (1 µL) of glycogen.
9. Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn).
10. Add 2X volume of EtOH.
    • Optional: incubate samples at -20°C for 10 min.
11. Precipitate DNA with a 10 min, 14K spin at 4°C. Wash pellet with 500 µL 75% EtOH.
12. Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8.
13. 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 (or NEB Buffer 2)
   • 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 (add 39.8 uL of mix per sample.)
   • 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. (Optional incubation at -20°C.)
5. Spin and wash with 200 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 10 mM Tris, pH 8.0.
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. (Optional incubation at -20°C.) 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.

Detection

• Continue with ChIP-chip or Gene-specific PCR to detect global or specific chromatin binding sites for your protein of interest.

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)

References

• ChIP protocol, Young Lab
• [1] Odom DT et al."Control of pancreas and liver gene expression by HNF transcription factors." Science. 2004 Feb 27;303(5662):1378-81. PMID: 14988562

Contacts

• cmc
• odom