Chromatin Immunoprecipitation (ChIP) Assay

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before. 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 (Brookfield, CT, United States). Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-MRTF-A (Santa Cruz, sc-10768), anti-SRF (Cell Signaling Tech, 5147, Danvers, MA, United States), anti-BRG1 (Abcam, ab110641, Cambridge, United Kingdom), anti-acetyl histone H3 (Millipore, 06-599, Burlington, MA, United States), and anti-trimethyl H3K4 (Millipore, 07-442, Burlington, MA, United States). Precipitated genomic DNA was amplified by real-time PCR with the following primers: ET1 proximal promoter, 5′-GGCGTCTGCCTCTGAAGT-3′ and 5′-GGGTAAACAGCTCCGACTT-3′. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as% recovery relative the input as previously described (Chen et al.,2020b,c (link)). All experiments were performed in triplicate wells and repeated three times.