title: GSM8714552: H3K9me3_JIB04_500nM_Rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-Seq experiments, 10 million OCI-Ly1 cells were fixed with 1% formaldehyde for 10 minutes at room temperature before termination with 0.125 M glycine. Cells were then lysed in ChIP buffer (0.6% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1) and cross-linked chromatin was sonicated to obtain DNA fragments of 300-800 bp. Chromatin was then centrifuged (10 min, 4°C, 13,000 rpm) and the supernatant was diluted 6-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, and 167 mM NaCl) with protease inhibitors (Roche Diagnostics). Diluted chromatin was incubated with 5 μg of primary antibody overnight at 4°C with agitation. Antibodies were pelleted using 50 μL of Dynabeads (ThermoFisher Scientific). The beads were sequentially washed once with the ChIP wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, and 150 mM NaCl), the ChIP wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, and 500 mM NaCl), the ChIP wash buffer 3 (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris pH 8), and finally the ChIP wash buffer 4 (10 mM Tris-HCl, 1 mM EDTA). Chromatin was eluted using ChIP Elution Buffer (1% SDS, 100mM NaHCO3). The cross-link between protein and DNA was reversed by heat incubation (65°C, overnight), and proteins were then digested with proteinase K. DNA was recovered by phenol-chloroform extraction and ethanol precipitation in presence of 20 µg of glycogen (Sigma Aldrich). ChIP-seq libraries were prepared using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB cat. no. E6040). Libraries were single-end indexed and sequenced on an Illumina HiSeq 2000 to generate 50 bp single end reads.