title: GSM9223653: BL6-SOX2-Control-1; Mus musculus; ChIP-Seq
library_construction_protocol: SOX2-ChIP analysis was performed as described previously with modifications. Briefly, 10 million cells were crosslinked with 2mM DSG in PBS for 50 min, followed by 1% formaldehyde for 10 min at room temperature, and quenched with 120mM Glycine for 10 min. The crosslinked cells were washed with ice-cold PBS, flash frozen, and stored at -80°C. For nuclear extraction, the cell pellet was thawed on ice, resuspended in lysis buffer (50mM HEPES-KOH [pH7.5], 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, Complete protease inhibitor cocktail) and lysed with a Dounce homogenizer. Nuclei were pelleted by centrifugation (1350g, 5min, 4°C), washed with wash buffer (10mM Tris-HCl [pH 8], 200mM NaCl, 1mM EDTA, 0.5mM EGTA, Complete protease inhibitor cocktail), and resuspended in sonication buffer (10mM Tris-HCl [pH 8], 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, Complete protease inhibitor cocktail). Nuclei were sonicated using a Covaris E220, and chromatin was centrifuged (20,000g, 10min, 4°C). Fragment size distribution and DNA content were assessed using a Nanodrop spectrophotometer and TapeStation. Chromatin and input samples were snap-frozen and stored at -80°C. For immunoprecipitation, 30μl Dyna Protein-G magnetic beads (Thermo Fisher Scientific) were washed with blocking solution (0.5% BSA in PBS-Tween-20), incubated with 10μg antibody (IgG or SOX2) and mixed with 40μg chromatin overnight at 4°C. Beads were washed with wash buffer (50mM HEPES-KOH pH7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and TE buffer (10mM Tris-HCl pH8, 1mM EDTA, 50mM NaCl). Bound chromatin was eluted with elution buffer (50mM Tris-HCl pH8, 10mM EDTA, 1% SDS) at 65°C for 30 min, and crosslinks were reversed by incubation at 65°C for 16 hr. RNA was digested with 0.2mg/ml RNase-A for 2 hr at 37°C, proteins with 0.2mg/ml Proteinase-K for 2 hr at 55°C. DNA was purified using Qiagen Qiaquick PCR cleanup columns, quantified by Qubit Fluorometric Quantitation, and sequenced on the Illumina Nextseq 2000 platform in paired-end mode with 150 bp per read. NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1)
identifiers.namespace: GEO
identifiers.id: GSM9223653_r1