title: GSM9052562: hlsec, shPML, Vehicle, PML(ChIP), rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells used for ChIP-seq were harvested and crosslinked with 1% formaldehyde in PBS, followed by quenching with 125 mM glycine on ice. The samples were washed with cold PBS and collected. The cells were then pelleted for 10 minutes at 4 °C, resuspended in lysis buffer I (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors), rotated for 10 minutes at 4 °C, then resuspended in lysis buffer II (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors). The cells were rotated once more for 10 minutes at 4 °C, and resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, and 1% Triton X-100, protease inhibitors) for sonication. Sonicated lysates were centrifuged for 10 minutes at 4 °C. The sonicated chromatin was then incubated with magnetic beads bound with PML antibody overnight at 4 °C to enrich for DNA. The beads were then washed with wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1mM EDTA pH 8.0, 0.7% Na-Deoxycholate, 1% NP-40) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl) in order. Chromatin elution was performed by incubation in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65°C for 30 minutes. Cross-links were reversed overnight at 65 °C. To remove protein, proteinase K was added and incubated at 37 °C for 2 hours. DNA was then purified, amplified via PCR, and sequenced on the Illumina platform.