Ab264350

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Wu et al., 2020 (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 phenylmethylsulfonyl fluoride (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-E2F5 (Thermo Fisher, PA5-81166), anti-TFDP1 (Thermo Fisher, PA5-86135), anti-acetyl H3 (Millipore, 06–599), anti-trimethyl H3K4 (Millipore, 17–614), anti-RNA polymerase II (Abcam, ab264350), anti-p300 (Abcam, ab275378), anti-KMT2A (Bethyl laboratories, A300-087A), or pre-immune IgG. Precipitated DNA was amplified with the following primers: 5′-AGGCTTTTCCCCGCCCTC-3′ and 5′-AGAGAGAGAGAGTCGGAAAAAAG-3′.

ChIPs were performed using an EZ-Magna ChIP Chromatin Immunoprecipitation Kit (Millipore, USA), as previously described [26 (link), 27 ]. Briefly, after treatments, cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Then, cell lysis supernatant was carefully removed and nuclear pellets were re-suspended in 0.5 ml of nuclear lysis buffer, then sonicated to create appropriately sized chromatin fragments. After centrifuged to remove insoluble materials, supernatant was transferred to clean microfuge tubes in 50 μl aliquots. Then 3 μg anti-RNA polymerase II (Pol II) antibody (ab264350, Abcam, USA), or normal IgG was added to each nuclear extract, and extracts were further incubated at 4◦C overnight. Nuclear extracts were later incubated with magnetic protein A/G beads for 2 h at 4◦C to capture protein/DNA complexes, then beads were sequentially washed with low salt buffer, high salt buffer, LiCl wash buffer and TE buffer, then protein/DNA complexes were eluted and reverse cross-linked to free the DNA. Purified DNA was analyzed by qPCR with primers listed in Table 1.