Chromatin Immunoprecipitation Assay Protocol

Chromatin immunoprecipitation assays were performed essentially as described before (Li et al., 2017 (link), 2018c (link),d (link), 2019b (link); Yu et al., 2017 (link), 2018 (link); Fan et al., 2019 (link); Kong et al., 2019a (link), b ; Liu et al., 2019b (link); Weng et al., 2019 (link); Yang et al., 2019a (link), b (link); Zhang et al., 2019 (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 ∼500 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with the following antibodies: anti-Brg1 (Santa Cruz, sc-17796), anti-acetyl histone H3 (Millipore, 06-599), anti-trimethyl H3K4 (Millipore, 07-473), anti-dimethyl H3K9 (Millipore, 07-441), anti-5′-hydroxymethylcytosine (Abcam, ab106918), anti-5′-methylcytosine (Abcam, ab10805), anti-TET1 (Active Motif, 61443), anti-TET2 (Millipore, MABE462), anti-TET3 (ABE290), anti-c-Jun (Santa Cruz, sc-1694), anti-c-Fos (Santa Cruz, sc-52), or IgG. Precipitated DNA was amplified with the following primers: for LGALS3 promoter (-402/-73), 5′-AATTTGTAGTCAGTTCCCTAG-3′ and 5′-AAATACTCCCAGCCCCGC-3; for LGALS3 promoter (-1276/-959), 5′-ATACCTGGTTTTCTCCATAG-3′ and 5′-ATATTGCCTATAAGCTACCC-3′.