ChIP assay: Chromatin immunoprecipitation assay was performed according to the protocol of ChIP assay kit (Upstate Biotechnology, Lake Placid, NY).
1, For cells cultured in 2D, about 1-2X107 S1 cells were growth in 100mm dish, and were cross-linked by adding formaldehyde to final concentration of 1% and incubated in room temperature for 10 minutes. For cells growth in 3D, cells were isolated from EHS using PBS/EDTA, and also cross-linked as cells growth in 2D.
2, Wash cells twice using ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1microgram/ml aprotinin and 1microgram/ml pepstatin A). Note: Add protease inhibitors to PBS just prior to use. PMSF has a half-life of approximately 30 minutes in aqueous solutions.
3, Scrape cells into conical tube, Pellet cells for 4 minutes at 2000 rpm at 4ºC.
4, Cells were washed with PBS and resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH8.0) add protease inhibitors (inhibitors: 1mM PMSF, 1microgram/ml aprotinin and 1microgram/ml pepstatin A).. After incubated 10 minutes on ice, cells were sonicated to shear DNA to lengths between 200 and 1000 basepairs being sure to keep samples ice cold.
5, Centrifuge samples (part A, step 7) for 10 minutes at 13,000 rpm at 4°C, and detected the OD260 of the lysates.
6, Sonicated lysates were then diluted to OD260 2 with ChIP dilution buffer (). 60 ul protein A-agarose beads (Upstate Catalog # 16-157) was added to sonicated lysates and rotated at 4 for one hour to reduce the non-specific dinding.
7, Pellet agarose by brief centrifugation and collect the supernatant fraction, 20ul of lystates were taken out as input control.
9, Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2ml supernatant fraction and incubate 2h at 4ºC with rotation. For a negative control, perform a no-antibody immunoprecipitation by incubating the supernatant fraction with 60 microliters of Salmon Sperm DNA/Protein A Agarose Slurry for one hour at 4ºC with rotation.
10, Pellet agarose by gentle centrifugation (700 to 1000 rpm at 4ºC, ~1min). Carefully remove the supernatant that contains unbound, non-specific DNA. Wash the protein A agarose/antibody/histone complex for 3-5 minutes on a rotating platform with 1ml of each of the buffers listed in the order as given below:
Low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 150 mM NaCl); High salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 500 mM NaCl); LiCl buffer (0.25 M LiCl, 1% NP-40, 1% SDC, 1 mM EDTA, 10 mM Tris-HCl pH8.0); TE buffer (20 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0).
11, Elute the histone complex from the antibody by adding 250 microliter elution buffer (1%SDS, 0.1M NaHCO3) to the pelleted protein A agarose/antibody/histone complex from step 10 above. Vortex briefly to mix and incubate at room temperature for 15 minutes with rotation. Spin down agarose, and carefully transfer the supernatant fraction (eluate) to another tube and repeat elution. Combine eluates (total volume=approximately 500 microliters.)
12, Add 20 microliters 5M NaCl to the combined eluates (500 microliters) and reverse histone-DNA crosslinks by heating at 65ºC for 4 hours.
Note: Include the input DNA from this step.
13, Add 10 microliters of 0.5M EDTA, 20 microliters 1M Tris-HCl, pH 6.5 and 2 microliters of 10mg/ml Proteinase K to the combined eluates and incubate for one hour at 45ºC.
14, Recover DNA by phenol/chloroform extraction and ethanol precipitation. Addition of an inert carrier, such as 20 micrograms glycogen, helps visualize the DNA pellet. Wash pellets with 70% ethanol and air dry.
15, Resuspend pellets in an appropriate buffer for PCR or slot-blot reactions. PCR or slot-blot conditions must be determined empirically.