title: GSM3983855: WT_ChIP.2; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin extracts were obtained by successive rounds of lysis in LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet-P40, 0.25% Triton X-100), LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine). Chromatin DNA was sheared to a size range of 100-500 bp by sonication. Chromatin immunoprecipitation was performed by incubating sonicated chromatin with anti-PRDM10 antibody at 4°C overnight, followed by incubation with Protein A Dynabeads. Beads were washed in low salt buffer, high salt buffer, LiCl buffer and TE buffer. Protein/DNA complexes were eluted, reverse-crosslinked overnight at 65°C and treated with RNase A and proteinase K. ChIP DNA was purified using Qiaquick PCR Purification kit (Qiagen). Libraries were prepared from 4 ng of ChIP DNA using the NEBNext Ultra II DNA Library Preparation Kit for Illumina (NEB). All libraries were amplified for 9 PCR cycles and final elution volumes were reduced. Libraries were quantified by High Sensitivity DNA Assay on a Bioanalyzer and quantitative real-time PCR (KAPA Library Quantification Kit for Illumina, Roche).