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 Cells were treated cells in the presence of 1α,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 1α,25(OH)2D3 (100 nM, 6h)