Chromatin Immunoprecipitation Analysis of H3K4me3 Levels

A673 cells were treated with vehicle (DMSO) or 2 μM JIB-04 for 36 hours. Chromatin immunoprecipitation analysis followed by qPCR (ChIP-qPCR), to compare H3K4me3 levels at the CDKN1A promoter in vehicle and drug-treated cells was performed essentially as previously described [18 (link)], with the following modifications: 4 x 10⁶ cells were resuspended in 500ul of lysis buffer, and sonicated in the Diagenode Bioruptor for 25 cycles (30sec on/90sec off on High setting); lysate was diluted up to 4x the volume before clearing with Protein A/G beads; 5 μg of control (rabbit IgG; Santa Cruz, sc-2027) and specific (H3K4me3; Invitrogen, #49-1005) antibodies were used for immunoprecipitation. Primers used for qPCR are listed in Supplementary Materials (Supplementary Table 2).