GSM8691061: PBS_Input_exp1; Mus musculus; ChIP-Seq
On day 4, 4x10^6 cells from each cell culture were collected, washed and fixed for 10 min in FBS-free RPMI containing 1% formaldehyde at RT and quenched with 0.125M glycine. Fixed cells were washed with ice-cold PBS, nuclei were prepared by cell lysis and chromatin shearing was performed by Covaris sonication. After sonication, an aliquot of sheared chromatin was kept as input. The rest of the sheared chromatin samples were proceeded to immunoprecipitation using antibodies specific for H3K9ac and PCAF. The immunocomplexes were isolated using magnetic beads and washed 6 times with RIPA buffer. DNA was isolated from immunoprecipitated complexes through proteinase K treatment, followed by reverse crosslinking at 65°C. DNA fragments were purified using NGS magnetic beads. ChIP-seq library construction and sequencing were carried out at the Greek Genome Center (GGC) of BRFAA. Libraries for H3K9ac and PCAF ChIPseq were generated with NEBNext Ultra II DNA Library Prep Kit for Illumina, as indicated by the manufacturer's guidelines (NEB, E7645L) using 10ng of DNA (chromatin that was not incubated with the antibody was used as input DNA). The quality of the generated libraries was validated with the Agilent bioanalyzer DNA1000 chip. Subsequently, the libraries were quantitated with the qubit HS spectrophotometric method and pooled in equimolar amounts for Sequencing.