ChIP Assay Protocol for Histone Modifications

ChIP assay was performed as we previously described^{15 (link)}. Briefly, cells were lysed by suspending in a sequence of lytic and purification buffers. DNA fragments of 300–800 bp long were obtained by treating the nuclear pellet, obtained from lytic buffers, with MNase (Cell Signaling, Beverly, MA) in digestion buffer for 6 min. DNA fragments were immunoprecipitated with specific antibodies (anti-trimethyl-Histone H3 Lys27 and rabbit IgG) at 4 °C overnight. Immunoprecipitated DNA fragments were extracted using protein-A sepharose (Sigma-Aldrich, St. Louis, MO) and purified using DNA purifying slurry (Diagenode, Denville, NJ). The amount of purified DNA was estimated by nanodrop of 200 ng purified DNA template using SYBR green chemistry as described in RT-qPCR quantification section. Promoter-specific primers used in the current study are described in Table 1. Calculations are expressed as a percentage of the input DNA.