GSM9001288: Asynchronous mouse embryonic fibroblasts batch 4, P3; Mus musculus; ChIP-Seq
Approximately 18 x 106 WT and KO MEFs synchronized at the G1/S phase by aphidicolin were allowed to progress into the S phase for 4 hours before they were harvested and washed with PBS. These S-phase-enriched cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature and quenched with 125 mM glycine at room temperature for 10 minutes while being rotated. The cells were pelleted, washed with cold PBS, and resuspended in cell swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, supplemented with protease and phosphatase inhibitors) on ice for 10 minutes to release nuclei. Nuclei were pelleted, resuspended in ChIP sonication buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors, and sonicated using a probe-based sonicator (FB120 with a CL-18 probe; ThermoFisher) at 25% amplitude with 30-second pulses at 30-second intervals for a total of 3 minutes. ChIP-seq libraries were prepared using the KAPA Hyperprep Library Kit (Roche Diagnostics). Normalization of the sample quantities was done before final amplification based on the quantification of the ligation products by qPCR. Library size distribution was assessed on a 2100 bioanalyzer (Agilent Technologies) using a High Sensitivity DNA Kit, and libraries were quantified by qPCR. Equimolar libraries were sequenced in paired-end readcounts (PE100) on a Novaseq system (Illumina) at 50 million readcounts per library.