title: GSM1619774: Input, untreated, replicate 2; Homo sapiens; ChIP-Seq
library_construction_protocol: MDA-MB-231 cells in culture were crosslinked using 1% formaldehyde for 10 minutes at room temperature, following which excess reagent was quenched by the addition of 125 mM glycine for 5 minutes. Cells were washed with ice cold PBS and the pellet was resuspended in ChIP lysis buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.1, protease inhibitor cocktail and PMSF). DNA was sheared to 200-500bp using a Branson probe sonicator while keeping the sample on ice. A small aliquot of sheared chromatin was saved as “input”. Chromatin from 2 million cells was used for each ChIP reaction and diluted 10-fold using ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, protease inhibitor cocktail and PMSF). Immunoprecipitation was performed by incubating diluted chromatin with Dynabeads G (Invitrogen, pre-washed with BSA/PBS) and the following antibodies overnight at 4°C: H3K9me2 (Abcam, ab1220; 2 mg per ChIP) or H3K9ac (Millipore #07-352; 3.5 ml per ChIP). Beads were subsequently washed using the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl buffer (0.25M LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and twice with TE (1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Dissociation of chromatin-antibody complexes from the beads was facilitated by eluting twice using an appropriate buffer (1% SDS, 0.1M NaHCO3) for 15 minutes each at RT. Crosslinks in all ChIP and input samples were reversed by incubating with NaCl overnight at 65°C. Samples were treated with RNAse A for one hour at 37°C followed by addition of Proteinase K for one hour at 45°C for cleanup. In the final step, DNA was purified using the MinElute PCR Purification Kit (Qiagen). Libraries were made using the Diagenode MicroPlex Library PreparationTM kit as per manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM1619774