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

To investigate the effect of 1,25(OH)2D3 activation on chromatin accessabilty 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, 4hr) or EtOH in triplicate independent experiments. Briefly, 50x103 cells were resuspended in 50ul of ATAC-resuspension buffer (ATAC-RSB - 10mM Tris-HCl, 10mM NaCl, 3mM MgCl2) containing (0.1% NP-40, 0.1% tween-20, and 0.01% digitonin) and pipetted up and down 3 times. Further, 1ml of ATAC-wash-resuspension buffer (ATAC-RSB + 0.1% tween 20) was used to pellet down the nuclei. The nucleic were further resuspended in transposition mix (2X TD buffer, 1X PBS, Digitonin 0.01%, tween 20 0.1%, NFW 5ul, and Illumina transposase 2.5ul). Mixing, cleanup and library preparation, quantification and sequencing was performed using NovaSeq6000 S1 PE150bp Sequencing as per protocol51 (28846090). (ATAC-Seq data were separated into nucleosome free (NF), mono-, di- and tri-nucleosome compartments (ATACSeqQC)52. Comparative chromatin accessability analsyes byATAC-seq analsyes of prostate cell lines treated with 1α,25(OH)2D3 (100 nM, 4h)