ChIP
From CCGB
CHROMATIN IMMUNOPRECIPITATION (ChIP)
-- by Yong Cheng
DAY 0
1. Culture 7.5×107 cells per IP
a. Set up 2 flasks with 150mL in each at a density of 0.5×106 cells/mL
b. Chemically induce differentiation in one flask (estradiol or tamoxifen for G1E; HMBA or DMSO for MEL; hydroxyurea, hemin, or sodium butyrate for K562)
DAY 1
2. Centrifuge cells at ~500×g for 5 minutes at 4°C in 50mL conical tubes
3. Remove all medium from tubes and resuspend cells in 200mL of PBS in an Erlenmeyer flask on a stir plate
4. Crosslink by adding formaldehyde to the gently-stirring cell suspension at a final concentration of 0.4% (2.162mL of 37%formaldehyde in a 200mL cell suspension). Continue stirring at room temperature for 10 minutes. Adjust the concentration of formaldehyde for different antibody can also work
5. To quench crosslinking reaction, add glycine to each flask at a final concentration of 125mM (1.9887g per flask). Continue to stir for 5 additional minutes at room temperature.
6. Collect crosslinked cell suspensions in 50mL conical tubes and centrifuge at ~500×g for 5 minutes at 4°C
7. Decant supernatant and resuspend cell pellets from each condition in a total of 1mL of cold PBS in a microcentrifuge tube
8. Centrifuge cell suspensions in microcentrifuge at 2000rpm for 5 minutes at 4°C
9. Aspirate supernatant and resuspend cell pellet in 500μL of COLD cell lysis buffer with PMSF and PI cocktail (CLB+) – add sodium butyrate if ChIPing for acetyl-histones
10. Allow cells to lyse on ice for 10 minutes
11. Centrifuge lysed cell solution in microcentrifuge at 2500rpm for 5 minutes at 4°C
11.1. resuspend the cells in 1ml PBS and transfer the cell to a 1.5 eppendorf tube.
12. Aspirate supernatant and resuspend nuclear pellet in 1mL of ROOM-TEMPERATURE nuclei lysis buffer with PMSF and PI cocktail (NLB+) – add sodium butyrate if ChIPing for acetyl-histones
13. Lyse nuclei for 10 minutes on ice
14. Add 600μL of COLD IP dilution buffer with PMSF and PI cocktail (IPDB+) – add sodium butyrate if ChIPing for acetyl-histones
15. Transfer lysed nuclear suspensions to Falcon 2059 tubes on ice
16. Sonicate suspensions on ice to shear chromatin Output power: 3 30 cycles of 1 second on, 1 second off sonication Do not let the tip touch the sidewall of the tube and insert the tip deep enough below the liquid surface, but do not touch the bottom. Misonix sonicator 3000 http://www.misonix.com/laboratory/Laboratory/Sonicator/Sonicator3000
17. Return sonicated suspensions to microcentrifuge tubes on ice and centrifuge in microcentrifuge at 2500rpm for 5 minutes at 4°C to pellet nuclear debris
18. Dilute supernatant into 3.4mL of COLD IPDB+ in a 15mL conical tube and keep on ice
19. Pre-clear chromatin overnight by adding 200μL of protein G agarose bead slurry and 20μg of appropriate non-immune sera and/or normal IgGs to each tube Protein G agarose (Invitrogen Cat No 15920-010) Rabbit IgG Upstate Anti-H3 ab Upstate Rabbit
Anti-H4 ab in serum Upstate Rabbit Rabbit serum ? (There are a lot of them in Santa Cruz) Gata1 (N6) Santa Cruz Rat IgG ER Ab-10(Clone TE111.5D11) neoMarkers
20. Allow chromatin solutions to rotate overnight in cold room
21. To pre-bind antibodies to protein G agarose beads, add, in microcentrifuge tubes on ice, for each IP condition, 10μg of IP antibody, 50μL of protein G agarose bead slurry, and 1mL of cold PBS
- If you are ChIPing for GATA-1 and ER, you would need 8 tubes for this step, labeled:
GATA-1 (-)
GATA-1 (+)
ER (-)
ER (+)
rat IgG (-) [ isotype control for GATA-1 antibody ]
rat IgG (+)
mouse IgG (-) [ isotype control for ER antibody ]
mouse IgG (+)
22. Rotate pre-binding solutions overnight in cold room
DAY 2
23. Centrifuge pre-cleared chromatin in swinging-bucket table-top centrifuge at 1000×g for 3 minutes at 4°C, then place on ice
24. Centrifuge pre-bound antibody:bead complexes in microcentrifuge at 7500rpm for 5 minutes at 4°C – aspirate supernatant, being sure not to disrupt the beads
25. Add 1mL of pre-cleared chromatin to each microcentrifuge tube of antibody:bead complex (IP samples)
26. Save 200μL of pre-cleared chromatin to use as input sample
27. Rotate IP samples in cold room for 2-4 hours
28. Centrifuge samples at 7500rpm for 5 minutes at 4°C in microcentrifuge
29. Let beads settle to bottom of tube on ice for 3-4 minutes
30. Aspirate supernatant, being sure not to disrupt the beads
31. Wash beads 6 times by addition of 500μL of cold buffer, brief vortexing, and repeating steps 28-30
- 1 time with COLD IP Wash Buffer I
- 2 times with COLD IP High Salt Wash Buffer
- 1 time with COLD IP Wash Buffer II
- 2 times with COLD TE Buffer
32. Elute DNA:protein complexes from beads TWICE with 100μL of freshly made room temperature elution buffer add water first when prepare the solution
33. Add 16μL of 5M NaCl to each of the 200μL eluates and the input samples (from step 26)
34. Add 1μL of 1mg/mL RNase A to each tube (including inputs) and incubate overnight at 65°C Rnase A Roche Cat No:10109169001
DAY 3
35. Add 3μL of 20mg/mL proteinase K solution to each tube (including inputs, again) and incubate for 2 hours at 45°C
36. Add 200μL of TE buffer to input and IP samples (for a final volume of 400μL)
37. Add 10μg of tRNA to each tube and immediately extract with phenol:chloroform:IAA
38. Extract input samples again with phenol:chloroform:IAA
39. Extract all samples once with chloroform
40. Add 5μg glycogen and 5μg tRNA to each input and IP sample
41. Add 2.5 volumes (1mL) of COLD 100% ethanol to each tube and mix well
42. Allow DNA to precipitate at -20°C for 30 minutes
43. Centrifuge at top speed (13000rpm) in microcentrifuge for 30 minutes at 4°C
44. Wash DNA pellet once with COLD 70% ethanol
45. Decant ethanol and air dry pellet for 10 minutes upside down, then 10-15 minutes in 37°C heat block or incubator
46. When all ethanol has evaporated, resuspend in 200μL of TE, pH8.0
For input, the concentration is 62ng/ul (by fluoremeter)
For the GATA1 ChIP DNA is 4 ng/ul
BUFFER RECIPES
CELL LYSIS BUFFER
10mM Tris, pH8.0
10mM NaCl
0.2% NP40 (IGEPAL)
NUCLEI LYSIS BUFFER
50mM Tris, pH8.1
10mM EDTA
1% SDS
IP DILUTION BUFFER
20mM Tris, pH8.1
2mM EDTA
150mM NaCl
1% Triton X-100
0.01% SDS
IP WASH BUFFER I
20mM Tris, pH8.1
2mM EDTA
50mM NaCl
1% Triton X-100
0.1% SDS
IP HIGH SALT WASH BUFFER
20mM Tris, pH8.1
2mM EDTA
500mM NaCl
1% Triton X-100
0.1% SDS add water first
IP WASH BUFFER II
10mM Tris, pH8.1
1mM EDTA
250mM LiCl
1% NP40 (IGEPAL)
1% Deoxycholic Acid
ELUTION BUFFER
100mM NaHCO3
1% SDS
