ChIP-Atlas: SRX2109207

Antigen

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: Cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched by glycine at a final concentration of 0.125 M, for 5 min at room temperature. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8, 0.1 % SDS, 10 mM EDTA pH 8, 1 mM PMSF, protease inhibitor cocktail) and chromatin was sonicated to an average size of 0.1-0.5 kb, using a Branson D250 sonifier (4 cycles of 30 seconds, 20% amplitude). 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).