GSE122572
BCL6 corepressor regulates androgen signaling and viability of prostate cancer cells
To analyze the role of BCOR (BCL6 corepressor) in androgen signaling, we used ChIP-seq to analyze chromatin binding of BCOR and positioning of H2AK119ub histone mark in androgen and vehicle treated VCaP cells. The role of BCOR in transcription regulation and positioning of H2AK119ub histone mark was analyzed from BCOR depleted (siBCOR) and control (siNON) treated VCaP cells in andorgen and vehicle treatments. Overall design: For ChIP-seq, VCaP cells were silenced using siRNA transfection for three days when indicate, grown on steroid-depleted medium for two days and treated for 1h with R1881 (10mM) or equal volume of vehicle (ethanol). Samples were fixed for 10 min. in room temperature with 1% (v/v) formaldehyde, fragmented to 200-400bp fragments by sonication (Bioruptor, Diagenode), chromatin immunoprecipitation was done using rabbit polyclonal anti-BCOR antibody (A310-672A, Bethyl Laboratories), antiserum against androgen receptor (AR) or anti-H2AK119ub antibody (#8240, Cell Signaling Technology), and libraries were prepared using NEBNext Ultra II kit (New England Biolabs). Two biological replicates of all conditions were sequenced using Illumina HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany). For RNA-seq, VCaP cells were transfected with siRNA against BCOR (siBCOR, 20nM, Dharmacon, ON-TARGETplus SMARTpool, L-004584-01-0005) or control siRNA (siNON, Dharmacon, non-targeting pool) using Lipofectamine RNAiMAX (Invitrogen). After three days, medium was changed to steroid-depleted medium for 32h, followed by treatment with dihydrotestosterone (DHT, 100nM) or equal volume of vehicle (ethanol) for 16h. RNA was extracted using RNeasy Plus Mini kit (Qiagen) and mRNA isolated using NEBNext Poly(a) mRNA Magnetic Isolation Module (New England Biolabs). Libraries were prepared using NEBNext Ultra II kit (New England Biolabs) and sequenced with HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany). Please note that the tdf processed data (for GSM4253755-GSM4253774 ChIP-seq samples) was generated from both replicates and is linked to the corresponding rep1 sample records