RARγ Cistrome Analysis in BAC-RARG-EGFP RWPE-1 Cells

ChIP was performed in BAC-RARG-EGFP containing RWPE-1 cells in the presence of CD437 (10 nM, 2 h) or DMSO as previously described [6 (link)]. Briefly, triplicate cultures of approximately 20 × 10⁶ cells were crosslinked with 1% formaldehyde solution, quenched with glycine (0.125 M) and harvested in cold PBS. Sonication of crosslinked chromatin was performed using a Bioruptor® UCD-200 TM Sonicator (Diagenode) for a total of 15 min. Immunoprecipitation of sonicated material was performed with antibodies against enhanced green fluorescent protein (ab290, Abcam) or IgG (sc-2027×, Santa Cruz) for 16 h, and antibody/bead complexes isolated with Magna ChIP^TM Protein A+G magnetic beads (Millipore). Complexes were washed, reverse crosslinked and treated sequentially with RNase and proteinase K prior to DNA isolation. Sequencing (100 bp single end, >30 × 10⁶ average reads/sample) was performed at the RPCCC Genomics Shared Resource core facility. The RARγ cistrome was analyzed with Rsubread/csaw [89 ], along with TF motif analyses (MotifDb). Binding site overlaps between RARγ and the other cistromes were tested (ChIPpeakAnno [90 (link)] and bedtools). Peak density plots were performed using the annotatePeaks.pl tool available from the HOMER (Hypergeometric Optimization of Motif EnRichment) suite.