GSM9290739: Control-H3K14ac_ChIPseq_Untreated; Mus musculus; ChIP-Seq
Cells were either cross-linked with 1% formaldehyde only (histone modifications, Fosb, Fosl2 ChIP-seq) for 10', RT or sequentially dual crosslinked (Brg1, PHF10 and ATF3 ChIP-seq) with 2mM DSG, 45', RT followed by 1% formaldehyde, 10', RT . Crosslinking was quenched with 125mM Glycine, 10', RT. The cells were washed by ice-cold 1X PBS, scraped and flash frozen to be stored at -80C until ready for processing. Cell pellets were thawed on ice, extracted with nuclear extraction buffer (50mM HEPES-KOH pH 8.0, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10' on ice and washed once with nuclear wash buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.5mM EGTA, 200mM NaCl). Nuclear pellets were resuspended in shearing buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.1% SDS) and sonicated using Branson SFX250 at 20% amplitude for 9 mins for dual cross-linked cells and 6 mins for single cross-linked cells at 0.5s ON and 1.5s OFF setting to obtain 200-800 bp fragment size. Sonicated chromatin was clarified by high-speed centrifugation and a 50ul aliquot was reverse cross-linked to check shearing efficiency. The chromatin was quantitated using absorbance at 260nm and further diluted with 2X ChIP dilution buffer (100mM HEPES-KOH pH 7.5, 600mM NaCl, 2mM EDTA, 2% Triton X-100, 0.2% Sodium Deoxycholate, 0.2% SDS) to obtain equal chromatin amounts in equal volume. ChIP-seq library preparation was done using Ovation Ultralow System V2 UDI according to manufacturer's directions and the final library was subjected to double sided size selection of 0.65x and 1x.