Chromatin Immunoprecipitation Sequencing Protocol

Cells were incubated with Hoechst 33342 (ThermoFisher Scientific H3570) at 1/1000 directly into the media for 1h at 37°C degrees. Cells were then trypsinized and resuspended in media containing Hoechst at 1/1000 before crosslinking with 1% formaldehyde for 13min at 37°C degrees and quenching with 0.125M glycine. Cells were washed with 1X PBS and resuspended in media containing Hoechst (1/1000). Cells were sorted with a BD FACS Fusion using the laser BV421-A based on DNA content. For siKDM5A ChIP, cells were harvested as previously described (10 (link),18 (link)). Sonication of chromatin was done with the Qsonica Q800R2 system (Qsonica). 0.5–10ug of chromatin were used based on DNA content (nanodrop concentrations) with the following antibodies: H3K4me1 (Abcam ab8895), H3K4me2 (Abcam ab32356), H3K4me3 (Millipore 07–473), H3K9me1 (Abcam ab8896–100), H3K9me2 (Abcam ab1220), H3K9me3 (Abcam ab8898). ChIP sequencing libraries were prepped using the TruSeq ChIP Sample Preparation kit (Illumina). Libraries were single-end sequenced (75 cycles) using a NextSeq500 (Illumina).