Chromatin Immunoprecipitation (ChIP) 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 et al.,2020a,b (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-Brg1 (Abcam, Ab110641), anti-hypoxia-inducible factor 1 alpha (HIF-1α) (Cell Signaling Techology, 14179), or pre-immune IgG. Precipitated DNA was amplified with the following primers: primer #1: 5′-ATATTACCAATCAGCGGCGAGC-3′ and 5′-ACTGCTATAGGGGGCGC-3′; primer #2, 5′-AAAAGCATGT GCCATTATGC-3′ and 5′-ATTCCTGGCTTTGTGGATGC-3′.