title: GSM2171230: H1299_T18_DMSO_H3K4; Homo sapiens; ChIP-Seq
library_construction_protocol: Drug treated H1299 Parental and H1299 T18 cells at 80% confluency (~10 million cells) were cross-linked with 1% formaldehyde for 10 minutes at 37°C, and quenched with 125 mM glycine at room temperature for 5 minutes. Following ice-cold PBS wash, the cell pellets were resuspended in 1.5 ml ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 2 x 15 minutes). The chromatin predominantly sheared to a fragment length of ~250 – 750 bp was centrifuged at 20,000 g for 15 minutes at 4°C. 100 μl of the supernatant was used for ChIP, and DNA purified from 30 μl of sheared chromatin was used as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4°C overnight with 10 μg of a mouse monoclonal anti-histone H3K27me3 antibody (Abcam, cat# ab6002) or a rabbit polyclonal anti-histone H3K4me3 antibody (Millipore, cat# 07-473). Immunoprecipitation was carried out by incubating with 40 μl pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl, protease inhibitors; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl, protease inhibitors; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 μl ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65°C in 192 mM NaCl for 16 hours. DNA fragments from immunoprecipitated chromatin and input were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 μl H2O according to the manufacturer’s protocol after treatment with RNase A and Proteinase K. Barcoded libraries of ChIP and input DNA were generated with the TruSeq® ChIP Sample Preparation Kit (Illumina).