title: GSM2831539: WT129_H3K9me3_ChIPSeq; Mus musculus; ChIP-Seq
library_construction_protocol: 5 million cells were fixed in growth media with 0.4% formaldehyde for 10 min and quenched with 125 mM glycine. Chromatin was released by sequential lysis with cells lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% Igepal) and nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS). Chromatin was sheared to ≈300 bp with 12 rounds of sonication (30 s on, 30 s off) on a Bioruptor (Diagenode) and incubated overnight at 4 ᵒC with either 5 μg H3K9me3 antibody (Abcam ab8898) or 10 μg H3K36me3 antibody (Abcam??). Samples were immunoprecipitated with Protein A Agarose beads (Sigma-Aldrich, 05015979001) and washed sequentially with low salt (20 mM Tris pH 8, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), high salt (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), and LiCl buffers (10 mM Tris pH 8, 1 mM EDTA, 0.25 M LiCl, 1% Igepal, 1% sodium deoxycholate). Chromatin was eluted with elution buffer (1% SDS, 100 mM NaHCO3), decrosslinked overnight at 65ᵒC, incubated with Proteinase K, phenol chloroform extracted and ethanol precipitated. Sample concentrations were determined by Qubit (ThermoFisher Scientific) and 20 ng of DNA was used as starting material. ChIP libraries were prepared with Nugen Ovation Ultralow System V2 (Nugen protocol M01379v1, 2014) with 10 cycles of amplification. Libraries quality was assessed by Qubit, Bioanalyzer (Agilent) and qPCR, and a single equimolar pool was made based on size adjusted qPCR quantitation. Following denaturation, 12 pM of library pools were used for cBot hybridisation and cluster generation (Illumina Protocol 15006165 v02 Feb 2016), and samples were sequenced on an Illumina HiSeq 1500 rapid mode (50 bp SR sequencing, Illumina Protocol 15035788 Rev D, Apr 2014).
attributes.tag: GEO Accession
attributes.value: GSM2831539