Chromatin Immunoprecipitation (ChIP) Assay Protocol

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Coarfa et al., 2020 (link); Hu et al., 2020 (link); Jehanno et al., 2020 (link); Maity et al., 2020 (link); Mallik et al., 2020 (link); Moon et al., 2020 (link); Shen et al., 2020 (link); Wang J. N. et al., 2020 (link); Wang S. et al., 2020 (link); Zhang et al., 2020 (link); Zhao et al., 2020 (link); Marti et al., 2021 (link); Peng et al., 2021 (link); Rashid et al., 2021 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-acetyl H3 (Millipore, 06-599), anti-trimethyl H3K4 (Millipore, 07-473), anti-trimethyl H3K9 (Diagenode, C15410193), anti-trimethyl H3K27 (Millipore, 04-449), anti-trimethyl H4K20 (Millipore, 07-463), anti-KDM4A (Abcam, ab191433), anti-KDM4B (Bethyl Laboratories, A301-477A), anti-KDM4C (Bethyl Laboratories, A300-885A), or anti-KDM4D (Proteintech, 22591-1-AP) antibodies.