Chromatin Immunoprecipitation (ChIP) Assay

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (38 (link),39 (link)). Briefly, 5 million cells were fixed with 1% formaldehyde and sonicated for 180 s (10 s on and 10 s off) on ice with a Branson sonicator with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin (1 ml) was clarified by centrifugation, aliquoted and snap-frozen in liquid nitrogen. To perform ChIP, sonicated chromatin (150 μl) was diluted 10-fold and purified with specific antiserum (2–5 μl) and protein G-agarose (60 μl). ChIP antibodies were obtained from Abcam (Cambridge, MA), including anti-trimethyl-H3-K4 (#ab8580), trimethyl-H3-K9 (#ab8898) and trimethyl-H3-K27 (#ab24684). DNA that was released from the bound chromatin after cross-linking reversal and proteinase K treatment was precipitated and diluted in 100 μl of low-TE buffer (1 mM Tris, 0.1 mM EDTA). PCR reactions were performed under liquid wax in a reaction containing 1 μl ChIP (or input) DNA, 0.5 μM appropriate primer pairs, 50 μM deoxynucleotide triphosphate and 0.2 U Klen-Taq I (Ab Peptides, MO). Standard PCR conditions were 95°C for 2 min, followed by 32 cycles of 95°C for 15 s, 65°C for 30 s of annealing and 72°C for 30 s of extension. The PCR products were separated on a 5% polyacrylamide–urea gel and quantified by a Phosphorimager (Molecular Dynamics, CA).