Characterisation of ER-beta functions in prostate cancer (ATAC-Seq)
To investigate the effect of a novel ER-beta selective ligand alone and with Enzalutamide on chromatin accessabilty in prostate cell lines Overall design: Cells were treated cells in the presence of OSU-ERb-12 alone (100 nM) or E2 alone (100nM), Enza (1 microMolar) or the combinations for 6h. 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). Comparative chromatin accessability analsyes by ATAC-seq analsyes of prostate cancer cells lines treated in triplicate with either vehicle, OSU-ERb-12 alone (100 nM) or E2 alone (100nM), Enza (1 microMolar) or the combinations for 6h