ChIP-qPCR protocol for Histone H3K27me3

ChIP assay was performed as described by Liu et al. [5 (link)]. Briefly, cells were lysed and treated with MNase (Cell Signaling, Beverly, MA, USA) to obtain DNA fragments of 300–800 bp that were then immunoprecipitated with target antibodies (anti-trimethyl-Histone H3 Lys27 and rabbit IgG) at 4 °C overnight. Extraction of immunoprecipitated DNA fragments using protein-A sepharose (Sigma-Aldrich) followed next prior to purification using DNA purifying slurry (Diagenode, Denville, NJ, USA). About 200 ng of purified DNA template was then amplified real-time PCR. Promoter-specific primers were used and are listed below (Table 3). Data was expressed as a percentage of the input DNA.