GSM5777951: PC9_AZD5153_2 [ATAC-seq]; Homo sapiens; ATAC-seq
Cells pellets were sent to Active Motif to perform the ChIP-seq assay using their standard protocol. Briefly, chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 µg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ul of antibody against H3K27ac (Active Motif cat# 39133, lot# 16119013). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR (qPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad).  The resulting signals were normalized for primer efficiency by carrying out qPCR for each primer pair using Input DNA. lllumina sequencing libraries were prepared from the ChIP and input DNA by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end).