Chromatin Immunoprecipitation

ChIP assays were done essentially as described (Boyd et al., 1998). Briefly, cells were crosslinked with 1% formaldehyde for 15 min at RT and collected by trypsinization and scraping. Cells were swelled in 5 mM Pipes (pH 8), 85 mM KCl, 0.5% NP40 in the presence of Complete Protease Inhibitor Cocktail (Roche), incubated on ice 20 min, and then dounced. Nuclei were collected and resuspended in IP buffer (150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl [pH 8], 1% Triton, and 0.1% SDS, Protease inhibitor). Samples were sonicated to an average length of 300–1,000 bp and then microfuged at 15,000 rpm for 1 hr. The chromatin solution was precleared by the addition of protein A sepharose blocked with 0.1ug/ml sonicated denatured salmon sperm DNA and 0.5 ug/ul BSA. Precleared chromatin was then incubated with 5 ul of anti-dimethyl  Lys-9 H3 antibody (Upstate Biotechnology) and rotated overnight at 4_C. Protein A sepharose, blocked as above, was added, and the incubation continued for 1 hr. Immunoprecipitates were washed twice with IP buffer, once with IP buffer/500 mM NaCl, once with 10 mM Tris-HCl (pH 8), 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA, and twice with TE. Immunoprecipitated material was eluted from the beads, and the DNA was recov-ered as described (Dudley et al., 1999). An aliquot of the chromatin was treated identically for use as the “Input.”