Protocol of Chromatin Immunoprecipitation (ChIP)

Chromatin immunoprecipitation (ChIP) assay involves cross-linking of proteins with DNA, fragmentation, and preparation of soluble chromatin followed by immunoprecipitation with an antibody recognizing the protein of interest. The segment of the genome associated with the protein is then identified by PCR amplification of the DNA in the immunoprecipitates. This is an abbreviated protocol to highlight the main points of ChIP. Refer to Troubleshooting of Chromatin Immunoprecipitation (ChIP) when you get in trouble.

Reagents

• Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS
• Formaldehyde
• Glycine
• Trypan blue
• PBS, pH7.2
• PBS containing 0.5 mM PMSF
• Swelling buffer: 25 mM HEPES, (pH 7.8), 1.5 mM MgCl₂, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, Protease inhibitor cocktail
• Sonication buffer: 50 mM HEPES (pH 7.9), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, Protease inhibitor cocktail
• Protein A or G agarose
• Antibody
• Wash buffer A: 50 mM HEPES (pH 7.9), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, Protease inhibitor cocktail
• Wash buffer B: 20 mM Tris (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 0.5 mM PMSF, Protease inhibitor cocktail
• TE buffer: 10 mM Tris HCl, 1.0 mM EDTA
• Elution buffer: 50 mM Tris (pH 8.0), 1 mM EDTA, 1% SDS, 50 mM NaHCO₃

Methods

This protocol is described for cultured cells grown in 150 mm dishes, containing 2-5 x 10⁷ cells per dish.

1. Replace medium with 27 mL DMEM containing 10% FBS.
2. Add 3 mL formaldehyde. Mix immediately. Incubate at RT for 10 minutes.

Note: Cross-linking time influences the efficiency of ChIP. For histone modifications, 10 minutes cross-linking is recommended. For transcription factors, longer cross-linking times can be employed (up to 30 minutes).

3. Add 3 mL glycine. Mix immediately.
4. Place the plate on the top of ice. Wash 3 times with 20 mL ice-cold PBS containing 0.5 mM PMSF.
5. Scrape cells in 20 mL ice-cold PBS containing 0.5 mM PMSF. Centrifuge at 1000 rpm for 5 minutes in cold centrifuge.

Note: Protease inhibitors are optional.

6. Resuspend pellet in at least 10 volume swelling buffer. Incubate in ice for 10 minutes. Dounce 10-20 times up-down. Check nuclei in microscope by mixing an aliquot with equal volume of 0.4% trypan blue.
7. Centrifuge at 2000 rpm for 5 minutes. Resuspend pellet (nuclei) in 5-10 mL sonication buffer.
8. Sonication. Keep sample in ice and allow sample to cool in ice for 1 minute between each sonication.

Note: It is very important to keep the sample cold during sonication. This step has to be optimized for each cell type and sonication instrument.

9. Centrifuge at 14000 rpm for 15 minutes.
10. Take the supernatant. Centrifuge again at 14000 rpm for 15 minutes.
11. Take the supernatant (the crude soluble chromatin). Add sonicated DNA (to 1 µg/mL final concentration) and BSA (to 1 mg/mL final concentration).
12. Preclear the lysate by incubating by constant rotation with Protein A or G agarose (use 40-50 µL agarose per mL lysate) for 2h in the cold room.
13. Centrifuge samples at 2000 rpm for 5 minutes. Remove the supernatant. This is the precleared soluble chromatin.
14. Save a 50 µL aliquot at -20°C. (For preparation of input DNA).
15. Divide the sample into 1 mL aliquots in microcentrifuge tubes for IP.
16. Add 5 µg of antibody. Rotate in the cold room for 2h. The concentration of the antibody should be empirically determined.
17. Add 40 µL Protein A or G agarose per IP (equilibrated as above). Incubate overnight by constant rotation in the cold room.
18. Centrifuge the beads at 6000 rpm for 3 minutes.
19. Wash 2 times with 1 mL sonication buffer. Each wash includes 10 minutes constant rotation of the tubes in the cold room. Wash 2 times with 1 mL wash buffer A. Wash 2 times with 1 mL wash buffer B. Wash 2 times with 1 mL TE buffer. Centrifuge at 6000 rpm for 3 minutes for each wash.
20. Add 200 µL elution buffer to the beads. Incubate at 65°C for 10 minutes. Centrifuge at 14000 rpm for 1 minute. Transfer supernatant to a new tube and elute beads again. Combine eluates and adjust with elution buffer if necessary.
21. Add 21 µL NaCl. Incubate at 65°C for at least 5h. This de-crosslinking step can also be done overnight.

Note: In parallel, thaw the input sample (50 µL) and supplement with 350 µL elution buffer. Add 21 µL NaCl.

22. Add 1 µL RNAse A. Incubate at 37°C for 1h. Add 4 µL EDTA and 2 µL Proteinase K. Incubate at 42°C for 2h.
23. Extract once with phenol/chloroform/isoamylalcohol and once with chloroform/isoamylalcohol.
24. Add 1 µL glycogen, 40 µL Na-acetate and 1 mL EtOH.
25. Vortex and leave to precipitate -20°C overnight.
26. Centrifuge at 14000 rpm for 30 minutes. Wash 1 time with 80% EtOH.
27. Resuspend IP and input samples in 100 µL 10 mM Tris (pH 7.5). Proceed to PCR analysis.

Procedure for Re-ChIP Assay

Re-ChIP stands for sequential ChIP with two antibodies to study the simultaneous presence of two proteins, or different modifications in the genome sequence of interest.

1. Wash cells 3 times with cold PBS (pH 8.0).
2. Prepare freshly 5 mM DTBP in ice cold PBS (pH 8.0) and add to plates sitting on ice. Incubate on ice for 30 minutes.
3. Wash cells twice with cold PBS (pH 8.0).
4. Add 20-25 mL ice-cold quenching buffer (100 mM Tris pH 8.0, 150 mM NaCl) per plate. Incubate on ice for 10 minutes.
5. Take the plates out from ice. Wash 3 times with PBS at RT.
6. Add 27 mL PBS to each plate + 3 mL formaldehyde. Mix well and incubate at RT for 10 minutes.
7. Continue with step 3 of standard protocol.