title: GSM2793934: NSCs_SETD5+/-_H3K27ac; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked adding formaldehyde directly to the cell culture media to reach a final concentration of 1%, then were incubated for 10’ at room temperature RT. The reaction was quenched adding glycine to a final concentration of 125 mM. Medium was then removed and cells were washed 3 times with cold, sterile PBS+protease inhibitor, then cells were gently scraped and collected for centrifugation at 4°C for 5’ at 1200 rpm. Five nanograms of immunoprecipitaded and purified DNA were used to generate ChIP-seq libraries. Briefly, end repair of DNA fragments was achieved by sequential 15 min incubations at 12°C and 25°C with T4 PNK (10U/μl), T4 POL (3U/μl) and 0.1 mM dNTPs. A-base addition was performed by incubating end-repaired DNA fragments with Klenow (3’ → 5’ exo, 5U/μl) and 167 μM dATP for 30 min at 30°C. Adaptor ligation was achieved by using the NEB Quick ligation kit (M2200L) and perfoming an incubation of 15 min at 25°C. Processed DNA fragments were finally amplified with a thermal cycler for 14 cicles, by using the Agilent PfuUltra II Fusion HS DNA Pol kit (600674). All DNA purification steps between the different enzymatic reactions were performed by using Agencourt AMPure XP SPRI beads (Beckman, A63882). The obtained libraries were subjected to quality control on Agilent Bioanalyzer (Agilent Technologies, G2943CA). ChIP-seq single-read 50bp