Chromatin Immunoprecipitation Protocol

Mirmira Lab ChIP Protocol

Reagents:

ChIP sonication Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mM Tris 8.1, 150 mM NaCl, 5 mM EDTA):
10 ml 10% Triton X-100
1 ml 10% Deoxycholate
5 ml 1 M Tris-Cl pH 8.1
1 ml 0.5 M EDTA
3 ml 5 M NaCl
80 ml Water
Just before use, add 10 ul Aprotinin, 10 ul Leupeptin, and 5 ul PMSF to each 10 ml.

High Salt Wash Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mM Tris-8.1, 500 mM NaCl, 5 mM EDTA)
10 ml 10% Triton X-100
1 ml 10% Deoxycholate
5 ml 1M Tris-8.1
1 ml 0.5M EDTA
10 ml 5M NaCl
73 ml Water

LiCl Immune Complex Wash Buffer

25 ml 1M LiCl
5 ml 10% IGEPAL
5 ml 10% Deoxycholate
1 ml 1M Tris-8.1
200 ul 0.5M EDTA
64 ml Water

Protease inhibitors (add 10 ul of each to 10 ml of PBS or sonication buffer)
Leupeptin 2 mg/ml in water
Aprotinin 2 mg/ml in water
PMSF 0.2 M

5 M NaCl    (29.22 gm/100 ml H2O)

1X TE Buffer (10mM Tris, 8.1, 1 mM EDTA)
(1 ml 1M Tris pH 8.1, 200 uL 0.5 M EDTA to 100 ml H2O)

0.5 M EDTA

1 M Tris-Cl, pH 6.8

Protein A/G Agarose (Santa Cruz #SC-2003)

Proteinase K (19 mg/ml, Roche Diagnostics # 03 115 828 001)

10X proteinase K buffer
(2.5 ml 1M Tris pH 7.5,  2.5 ml 0.5M EDTA, 12.5 ml 10% SDS, 
H2O to 25 ml)

Elution Buffer (1% SDS, 0.1 M NaHCO3, 0.01 mg/ml Herring sperm DNA, 2ng/ml CMV beta Gal plasmid)
(1 gm SDS, 0.84 gm NaHCO3, 100 ul of 10 mg/ml Sheared Herring
Sperm  DNA, 200 ng CMV Beta Gal plasmid DNA, to 100 ml H20)

10 mg/ml Herring Sperm DNA (Sigma # D3159)

37% Formaldehyde  (Fisher # F79-500)

1.25 M glycine  (9.38 gm in 100 ml H2O)

Phenol/ChCl3/Isoamyl Alcohol  (Fisher # BP1752-400)

10 mg/ml BSA (New England Biolabs # B9001S)

Protocol:  Generalized for many cell types, but may require optimization for specific cell types (tested with mPAC, bTC3, aTC, HEK, HeLa, NIH3T3).

For all the following steps, use the pipets that are specifically designated for ChIP use only.

Day 1:

1. To each 10 cm dish of cells, wash plate once with 10ml of PBS, then add 10 ml of Fresh PBS and add 270 ul of 37% formaldehyde, swirl gently to mix, and place at room temp 10 min.
2. At the end of the incubation, add 1 ml of 1.25 M glycine, swirl to mix.
3. Aspirate medium
4. Wash plate with 10 ml cold PBS x 2.  Aspirate PBS completely after the second wash.
5. Add 500 ul of cold PBS + protease inhibitors and scrape cells, collect in a 1.5 ml centrifuge tube.  At this point you should pool three plates worth of cells together in the same tube (I suggest using a 2 ml siliconized eppendorf tubes for this purpose).
6. Centrifuge at 2000 rpm for 2 min at 4 º C.
7. Remove and discard PBS
8. Add 600 ul of ChIP sonication buffer + protease inhibitors, and resuspend pellet (you can vortex vigorously at this point). 
9. Place on ice for 10 min.
10. Sonicate:  We use a Misonix Sonicator (model S-300 sonicator with 2.5 in diameter cup horn and 8-place sample holder for sonicating multiple samples).  The cup horn should be filled with an ice-water mixture.  We recommend the following sonication setting:
    
    Amplitude setting:  4
    15 five second pulses with 15 second cool-down intervals between pulses.  The shearing may be more efficient if the tubes can be placed at a 30-45 degree angle.
    Alternatively, you can use a setting of 4 and deliver two 90 sec pulses with 1 min 45 s cool-down interval between pulses. 
    We found that the two protocols are very similar in shearing DNA to the 500-2000 bp range, however, the first protocol minimizes heat build-up

11. Centrifuge at maximal setting at 4 C for 10-15 min.
12. Remove the supernatant into a fresh siliconized 1.5 ml eppendorf tube.  This is the Whole Cell Extract (WCE), and can be stored at –80 C at this point, if desired.
13. Check protein levels by making a 1:10 dilution of a sample of the extract in water and doing a Bradford assay.  Use 100 ug of WCE per antibody immunoprecipitation.  If not enough extract is available, bring the solution up to 100 ug protein by adding purified/acetylated BSA.
14. Pipet 100 ug WCE into a fresh siliconized tube containing ChIP sonication buffer with protease inhibitors to a final volume of 1 ml.
15. Pipet 10% of the WCE (input) into a 1.5 ml eppendorf tube (not siliconized, because it will dry faster after 70% EtOH wash—see later), and store at -20 C—you will need it later.
16. Add appropriate antibody volumes to each sample:
1. 5 ul for histone antibodies from Upstate (acH3, acH4, 2meK4, 3meK4)
2. 10 ul for histone antibody 1meK4
3. 5 ul for Pdx-1 rabbit polyclonal antiserum, RNA polymerase antibody from Santa Cruz
4. 2 ul for CTD RNA polymerase Ab
5. 10 ul for RNA polymerase Ser2 and Ser5 Ab
17. Place the samples on a nutator in the cold room, and incubate overnight

Day 2:

18. Resuspend Protein A agarose so that it forms a uniform suspension.  Using a pipet tip with the end clipped off, add 45 ul of this suspension to each immunoprecipitation.  Resuspend the protein A agarose each time before adding to the next sample, as it settles quickly.
19. Add 2 ul of a 10 mg/ml solution of herring sperm DNA
20. Place back on the nutator at 4 C for 1-2 h.
21. Centrifuge the samples at 4 C for 2 min at 2600 rpm.
22. Carefully remove the supernatant using a P-1000 and place it in a tube and label it “sample X—sup.”  Place this at –20 C in case you need it later.
23. Add 1 ml of COLD ChIP buffer (no protease inhibitors), invert the sample to resuspend the resin, and centrifuge for 2 min. at 2600 rpm.
24. Remove and discard the supernatant.
25. Wash X1 with COLD High Salt buffer, then X1 with COLD LiCl buffer, and then X1 with COLD TE
26. Add 250 ul of Elution buffer to the resin, and place on a nutator at room temp. for 15-20 min.
27. Centrifuge at top speed to pellet the resin, remove the supernatant to a fresh tube.
28. Repeat the elution step (step 26), and combine the supernatants. (you can now discard the tube containing the resin pellet)
29. At this time, add 500 ul of elution buffer to the “10% input samples” from step 15.
30. Add 20 ul of 5 M NaCl to each sample, vortex to mix, and place in a 65 C bath for 3-4 h.
31. Add 1 ml of ROOM TEMP ethanol to each sample place at –20 C overnight

Day 3:

32. Next day, spin the samples at top speed at 4 C for 15-20 min. to pellet the precipitated protein/DNA.
33. Aspirate off the supernatant, add 1 ml of ice cold 70% ethanol, spin again at 4 C for 5 min.
34. Aspirate off the sup, allow to air dry for 5-10 min.
35. Dissolve the pellet in 100 ul of TE.
36. Add 11 ul of 10X Proteinase K buffer, and 1 ul of a 19 mg/ml proteinase K solution and incubate at 55C for 1 hour.
37. Add 390 ul TE
38. Add 500 ul of PCIAA (phenol/chloroform/isoamyl alcohol)
39. Vortex for 30-60 sec and spin at max speed for 1 min.
40. Carefully remove the aqueous phase and transfer to a new tube.
41. Add 20 ul of 5 M NaCl, vortex to mix
42. Add 1 ml of room temp 100% EtOH
43. Incubate at -20 C overnight or -80 C for 1 h
44. Centrifuge at max speed for 20 min. at 4 C
45. Remove supernatant and add 1 ml of cold 70% EtOH
46. Centrifuge at max speed for 5 min at 4 C
47. Air dry tube
48. Resuspend in 100 ul of TE (Note:  you may NOT “see” a pellet, but that’s OK!)
49. You’re ready for PCR.