title: GSM6281915: MDA-MB-453_AR_input; Homo sapiens; ChIP-Seq
library_construction_protocol: Treated cells were cross-linked with 1% formaldehyde for 10 min, quenched for 5 min with 0.1 M glycine (pH 7.5), washed briefly, and scraped into ice-cold DPBS supplemented with 1x protease inhibitor cocktail (Roche). Cell pellets were resuspended in LB1 cytoplasmic lysis 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) at 4°C for 10 min. Nuclei were washed in LB2 buffer (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) at 4°C for 5 min, and subsequently resuspended in LB3 nuclear lysis buffer (10 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodiumdeoxycholate, 0.5% N-lauroylsarcosine). Lysates were sonicated using a Bioruptor Plus (Diagenode) to produce chromatin fragments of 200-1,000 bp. Trixon X-100 was spiked into sonicated lysates to a final of 1%, lysates were cleared of debris, and 5% of the sonicated chromatin was collected for an input control. Chromatin samples were immunoprecipitated overnight at 4°C with 50 µL of Protein A Dynabeads (Invitrogen) coupled to 2-5 µg of antibody. Three independent replicate experiments were performed, representing consecutive passages of cells. DNA was purified using phenol:chloroform:isoamyl alcohol extraction. 5 – 10 ng of ChIP DNA, along with pooled input chromatin collected from all replicates, was provided to the South Australian Genomics Centre for library preparation and sequencing. Recovered DNA was amplified using the Ultra Low Library Prep Kit (Qiagen) and libraries were sequenced on the Illumina NextSeq 500 to a depth of a minimum of approx. 20 million  75 bp single-end reads per sample. ChIP-Seq; Illumina NextSeq 500 (High Output v2) single-end 75 bp reads.