title: GSM9269366: D0_WT_H3K4me2_repA; Mus musculus; ChIP-Seq
library_construction_protocol: Approximately 10 million and 40 million cells were used for histone PTMs and COMPASS subunits ChIPs, respectively.  For histone modification ChIP assays, cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature (RT) before being quenched with 0.125 M glycine for another 10 minutes, while for COMPASS subunits ChIP assays cells were double crosslinked with ChIP cross-link Gold (Diagenode, C01019027) and 1% formaldehyde according to the manufactor's instructions. Chromatin was isolated through sequential incubation with three distinct lysis buffers: LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 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 sonication was carried out in LB3 using an EpiShearTM sonicator (Active Motif; 4-5 minutes, 20 seconds ON and 30 seconds OFF at 25% amplitude). The sheared chromatin was then incubated O/N at 4 °C with either 5 or 10 μg of antibody for histone PTMs or COMPASS subunits respectively. DNA libraries were prepared using the TruSeq ChIP-seq sample preparation kit. 10 ng of ChIP DNA was used as starting material.