GSM8782016: LNCaP.42D.PN.FBS.10uMENZA_AR-N_rep1; Homo sapiens; ChIP-Seq
Chromatin isolation, immunoprecipitation and sequencing. Cells were seeded in 15 cm² dishes in their respective growth medium and cultured for 3 days before performing ChIP experiments. Cells were fixed with 1% formaldehyde at room temperature for 10 minutes, and then quenched with 125 mM glycine. Cell nuclei were sequentially isolated using LB1 buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) followed by LB2 buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA). The nu-clei were then lysed with ChIP lysis buffer (1% SDS, 5 mM EDTA, and 50 mM Tris-HCl, pH 8.1). Chromatin was sheared into ~300 bp fragments using the Bioruptor Plus Sonicator (Diagenode). Immunoprecipitation was performed using an anti-AR antibody (ab108341). The precipitated protein-DNA complexes were reverse cross-linked overnight at 65°C. The following day, both input and ChIP DNA were purified using the QIAquick PCR Purification Kit (Qiagen, 28104). ChIP-seq libraries were prepared using the ThruPLEX DNA-Seq Prep Kit (Takara Bio, R400675). Next-generation sequencing (100 bp, paired-end) was conducted on an Illumina NovaSeq 6000 at the Fred Hutch Genomics Core.