title: GSM4483258: H3K27ac ChIP-seq from Luminal1/FOXA1 k.o tumor; Homo sapiens; ChIP-Seq
library_construction_protocol: We crosslinked 1–2 × 106 human bladder tumor cells for 5 min at 37 °C with formaldehyde at a final 1%. We then carried out ChIP using previously described methods.80 In brief, we pre-cleared sonicated chromatin in RIPA buffer (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS) with 2 μg of rabbit IgG (Cell Signaling Technology) and 20 μl of Protein G magnetic beads (Thermo Fisher Scientific). We then immunoprecipitated pre-cleared chromatin using 2 μg of H3K27ac antibody (Abcam) and 20 μl of Protein G magnetic beads (Thermo Fisher Scientific) overnight at 4 °C with rotation. The next day, we washed the pulled chromatin, and we reverse crosslinked it with proteinase K (20 mg/ml) and 10% SDS overnight at 65 °C. We extracted ChIPed DNA with phenol/chloroform and eluted it in 20 μl of TE. We constructed ChIP-seq libraries using the ACCEL-NGS® 2S PLUS DNA LIBRARY KIT (Swift Bioscience), following the manufacturer's instructions. In brief, purified ChIP DNA was subjected to end-repairing and adaptor ligation, and applied 12 cycles of PCR amplification. Amplified DNA was size selected and quantified using Bioanalyzer (Agilent). We sequenced ChIP-seq libraries using an Illumina HiSeq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM4483258