title: GSM2256184: input_u263_2; Homo sapiens; ChIP-Seq
library_construction_protocol: Genome-wide histone modifications were determined by ChIP against H3K9me3 (Abcam ab8898) on 5 million cells as described in (14). Cells were cross-linked by adding 37% formaldehyde to a final concentration of 1% into culture medium and gently shaking for 10 min at room temperature. Reaction was quenched with glycine, and cells were then washed twice with ice-cold PBS containing protease inhibitors (1mM PMSF, 1X Roche cOmplete EDTA-free cocktail). Cells were scraped off of the plate using a cell lifter and pelleted for 5 min at 2,000 rpm at 4°C. Pellet was snap-frozen in liquid nitrogen and stored at −80°C. Pellet was then thawed and resuspended in Cell Lysis Buffer (5 mM PIPES pH 8, 85 mM KCl, freshly added 1% IGEPAL) with protease inhibitors (Pierce Halt Protease Inhibitor Cocktail). Cells were then homogenized using a type B glass dounce homogenizer, pelleted, and resuspended in Nuclei Lysis Buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS). Chromatin was sonicated in Diagenode TPX tubes using the Diagenode Bioruptor for 20 cycles and DNA was ranged from 150–700 bps as determined by gel electrophoresis. Debris was pelleted and discarded, and an aliquot was removed for Input DNA sequencing from the sonicated chromatin within the supernatant. Sonicated chromatin was then diluted 5-fold in IP Dilution Buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% deoxycholic acid, 1 mM EDTA pH 8) with protease inhibitors and pre-cleared with Life Technologies Protein G Dynabeads for 2 hr at 4°C. 5 μg of antibody was added per million cells, and samples were incubated overnight at 4°C. Antibody-bound chromatin was then collected using Life Technologies Protein G Dynabeads and washed twice with IP Dilution Buffer, twice with IP Wash Buffer 2 (100 mM Tris–HCl pH 9, 500 mM LiCl, 1% IGEPAL, 1% deoxycholic acid), and once with IP Wash Buffer 3 (100 mM Tris–HCl pH 9, 500 mM LiCl, 150 mM NaCl, 1% IGEPAL, 1% deoxycholic acid). Precipitated chromatin was then eluted for 30 min at 65°C with Elution Buffer (1% SDS, 50 mM NaHCO3). ChIP and Input DNA crosslinks were reversed by adding 5 M NaCl and heating at 65°C overnight. The following day, 10 mg/ml RNase A was added to precipitated chromatin, and chromatin was incubated for 30 min at 37°C. DNA was then recovered using Agencourt AMPure XP Beads and quantified using the Life Technologies Qubit Fluorometer. Kapa HyperPlus