title: GSM3901404: CSB-H2O2 H3K9me3 input; Homo sapiens; ChIP-Seq
library_construction_protocol: CSB cells were cross-linked in a solution of 1% formaldehyde in PBS for 10 min at room temperature. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. The cells were harvested and washed three times with cold PBS, and cytosolic fractions were eliminated with buffer A (5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% NP-40, protease inhibitor cocktail (GenDEPOT, Katy, TX, USA)). Nuclear pellets were resuspended in buffer B (100 mM Tris-Cl (pH 8.1), 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, protease inhibitor cocktail), and the chromatin was sheared with S-450 sonicator (Branson, Danbury, CT, USA). Sonication condition was optimized by analyzing purified DNA samples with BIOANALYZER (Agilent, DNA1000 Kit)The prepared chromatin fraction (500 μg total sheared DNA per sample) was diluted 1/10 in IP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl (pH 8.1), 167 mM NaCl and a protease inhibitor cocktail) and incubated with antibodies, overnight at 4 °C. Samples were incubated for 2–4 h at 4 °C with protein A or G beads. Then the beads were washed with TSE150 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl (pH 8.1), 150 mM NaCl), TSE500 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl (pH 8.1), 500 mM NaCl), Buffer III (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-Cl (pH 8.1)) and two times with TE (pH 8.0) for 10 min in each solution. Bead-bound chromatin was eluted with elution buffer (1% SDS, 0.1 M NaHCO3 (pH 8.0)) for 1-2 hours at 65.5 °C. The supernatant containing the chromatin, without the beads, was isolated and incubated overnight at 65 °C with 200 mM NaCl to reverse cross-linking. Five hundred microliters of the sample were incubated at 50 °C after adding 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris (pH 6.5) and 4 μl of Proteinase K (20 mg ml−1) and then purified with phenol/chloroform/isoamyl alcohol. Nucleic acids were precipitated by centrifugation for 30 min at 4 °C after mixing the sample with 1 μl of glycogen solution (20 mg ml−1), 20 μl 5 M NaCl and 500 μl isopropanol. Purified nucleic acid pellets were washed with 70% ethanol, dried and dissolved in nuclease-free water. According to the manufacturer's protocol
attributes.tag: GEO Accession
attributes.value: GSM3901404