Characterisation of VDR signaling in prostate cancer health disparities (ChIP-Seq)

To investigate the effect of 1,25(OH)2D3 activation on VDR genomic binding in prostate cell lines derived from European Americans and African Americans Overall design: Cells were treated cells in the presence of 1a,25(OH)2D3 2D3 (100nM, 6hr) or EtOH in triplicate independent experiments. Briefly, approximately 20x106 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-200TM Sonicator (Diagenode) with optimized cycles for each cell type. Immunoprecipitation of sonicated material was performed with antibodies against VDR (D2K6W) – Cell Signaling) or IgG (Rabbit IgG sc2729 – Santa Cruz) for 16 hours, and antibody/bead complexes isolated with Magna ChIPTM Protein A+G magnetic beads (Millipore). Complexes were washed, reverse crosslinked, and treated sequentially with RNase and proteinase K prior to DNA isolation. Comparative VDR cistrome analsyes by ChIP-seq analsyes of prostate cell lines treated with 1a,25(OH)2D3 (100 nM, 6h)