GSM7867646: MCF-7, ERa, ChIP, GDC-9545 rep 1; Homo sapiens; ChIP-Seq
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. An aliquot of chromatin (30 ug) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using relevant antibodies against specified targets. 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. The ChIP reactions also contained a drosophila chromatin spike in for the normalization of the sequencing data. ChIPseq was performed by Active Motif.