title: GSM7207926: H3K27ac-L-E145-Col2a1GFP-GFPn-R1; Mus musculus; ChIP-Seq
library_name: GSM7207926
library_construction_protocol: Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a E220 focused-ultrasonicator (Covaris). Following FACS sorting, cells were centrifuged for 5′ at 400 × g at 4 °C and supernatant was discarded. Cells for ChIP-seq were resuspended in 10% FCS/PBS and fixed in 1% formaldehyde. The fixation was blocked by the addition of 1.25 M glycine, cells were isolated by centrifugation (1000 × g, at 4 °C for 8'), resuspended in cold lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, Protease Inhibitor (Roche, 04693159001)) and incubated on ice for 10' to isolate the cell nuclei. The nuclei were isolated by centrifugation (1000 × g, at 4 °C for 3′), washed in cold 1× PBS, and stored frozen at −80 °C. 5 × 105 fixed nuclei were sonicated to a 200–500 bp length with the Bioruptor Pico sonicator (Diagenode). H3K27Ac ChIP (Diagenode, C15410174) was performed as previously described (68), using 1/500 dilution of the antibody, with the addition of 5 mM of Na-Butyrate to all buffers. Libraries were prepared with <10 ng quantities of ChIP-enriched DNA as starting material and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Libraries were sequenced on an Illumina HiSeq 4000 with single-end reads of 50 or 100 bases.