GSM8898292: Old_p65_ChIP; Mus musculus; ChIP-Seq
30 million cells were fixed with 1/250 of Gold fixative (C01019027, Diagenode) for 30 minutes, and later a second fixation with 1% methanol-free formaldehyde was performed for 10 minutes. After quenching and cell lysis, nuclear extract was sonicated using a Bioruptor Pico (Diagenode). For IP, 30ug of chromatin were used and incubated with 30 ul of anti-YAP (CS14074, Cell Signaling Technology) with rotation overnight at 4ºC. After immunoprecipitated chromatin was eluted, DNA was purified with the QIAquick PCR Purification Kit (28106, Qiagen). Libraries were carried out using the NEBNext Ultra DNA Library Prep for Illumina kit (E7370) or the NEBNext® Ultra II DNA Library Prep for Illumina® kit (E7645) using the manufacturer's protocol. In brief, ChIP-enriched DNA and input DNA were subjected to end repair and the addition of “A” bases to 3' ends, NEB adapter's ligation and USER excision. For all purification steps, AgenCourt AMPure XP beads (A63882, Beckman Coulter) were used. Library amplification was performed by PCR using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, E6440, E6442, E6444, E6446). Analysis of final libraries was carried out using either an Agilent Bioanalyzer or Fragment analyzer High Sensitivity assay (5067‐4626 or DNF‐474) to estimate the quantity and check size distribution. Quantification of these final libraries was done by quantitative PCR using the KAPA Library Quantification Kit (KK4835, KapaBiosystems, 07960204001 Roche).