2.7. Chromatin Immunoprecipitation (ChIP) analysis

Chromatin Immunoprecipitation (ChIP) analyses were performed using EZ-ChIP kit according to manufacturer’s protocol (Millipore). Briefly, cells were cross-linked with paraformaldehyde, lysed and chromatin were prepared by sonication. Immunoprecipitation of chromatins were performed using antibodies specific to H3R8me2s (Epigentek- Cat # A-3706), H4R3me2s (Sigma aldrich- Cat # SAB4300870). DNA were purified from the immunoprecipiated chromatin and subsequently analyzed by qPCR using promoter specific primers (Supplementary table 2). Data were analyzed following percent input method as described below. ΔCt [normalized ChIP] = (Ct [ChIP] - (Ct [Input] - Log2 (Input dilution factor))), where Input Dilution Factor = (fraction of the input chromatin saved)⁻¹. In our analyses, 1/100th of the chromatin used for immunoprecipitation was used as input. Finally, the percentage (Input %) value for each sample is calculated as follows: Input % = 100/2 ^{ΔCt [normalized ChIP]}. The “Input %” value represents the enrichment of certain histone modification on specific region. Three independent biological replicates were analyzed.