GSM8862411: LNCaP_sgPTGES3, biological replicate 2, technical replicate 1 (ChIP-AR); Homo sapiens; ChIP-Seq
Fixation was carried out with 1% formaldehyde for 10 minutes at room temperature and subsequently quenched by adding 125 mM glycine. Nuclei were sequentially isolated using ice-cold LB1 buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), followed by ice-cold LB2 buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Subsequently, nuclear lysis was performed on ice for 10 minutes using ChIP lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 8.1). Chromatin was sheared to approximately 300 bp fragments with a Bioruptor Plus Sonicator (Diagenode). Immunoprecipitation was carried out using an anti-AR antibody (ab108341). Protein-DNA complexes were reverse cross-linked overnight at 65°C. ChIP and input DNA samples were purified the following day with the QIAquick PCR Purification Kit (Qiagen, 28104). Sequencing libraries were constructed from approximately 0.8 ng ChIP DNA using the ThruPLEX DNA-Seq Prep Kit (Takara Bio, R400675) with 12 cycles of PCR amplification.