GSM4040992: InputDNA_MajorAlleleHom1_rep2; Homo sapiens; ChIP-Seq
Cells were harvested, counted and resuspended in fresh cell culture media (10^6 cells/ml). For cross-linking, 37% Formaldehyde was added to a final concentration of 1%, and cells were placed on a rocker for 10 minutes (room temperature). Cross-linking was quenched by adding Glycine (final concentration 0.125M) and shaking for 5 min (room temperature). Cells were washed twice in ice cold PBS and cell pellets were lysed for 10 min at a density of 10^7 cells/ml in lysis buffer supplemented with Protease inhibitor (Complete Mini EDTA-free Protease Inhibitor cocktail tablets; Roche). Lysis buffer: 50mM HEPES pH 7.9, 140mM NaCl, 1mM EDTA pH 8.0, 10% v/v Glycerol, 0.5% v/v IGEPAL CA-630, 0.25% v/v Triton X-100. Lysates were washed twice with wash buffer (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0) and nuclei were prepared by washing twice with shearing buffer (0.1% w/v SDS, 1mM EDTA, 10mM Tris-HCl pH 8.0). Nuclei were resuspended in 200µl shearing buffer per 10^7 cells and sonicated for 9 cycles (30s ON/30s OFF) using a Bioruptor Pico. Triton X-100 and NaCl were added to the sheared chromatin to a final concentration of 1% and 100mM, respectively. 10µg of sheared chromatin were cleared by centrifugation (21,000G, 10min, 4C) and immunoprecipitation of histone-DNA complexes was performed overnight at 4ºC with rotation using an anti-H3K27ac antibody and the SimpleChIP Plus Sonication ChIP kit (Cell Signaling Technology). 50ng of immunoprecipitated DNA or input DNA were used to prepare sequencing libraries using the iDeal Library Preparation kit (Diagenode), according to manufacturer instructions. 10 PCR cycles were used for the amplification step and size selection was not performed. The quality and molarity of all libraries was assessed using a BioAnalyzer 2100 (Agilent) and the libraries were sequenced in pools of 8, with each pool being sequenced in 2 lanes of an Illumina HiSeq2500 high output flow-cell (50bp, single-end reads)