GSM9155196: THP1,Compound49,24h,ChIP-seq,H3K27ac; Homo sapiens; ChIP-Seq
cells were fixed with 1% formaldehyde for 15 minutes and quenched with 0.125-M glycine. Chromatin was isolated by adding lysis buffer, then disrupted with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300 to 500 bp with the Active Motif EpiShear probe sonicator (#53051) and cooled sonication platform (53080). 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 20 µL of antibody against PU-1 (Santa Cruz, sc-352), or 4 µL of antibody against H3K27Ac (Active Motif, 39133). Illumina libraries were prepared by Active Motif using standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation according to the standard Illumina ChIP-seq library.