GSM5388446: Tamoxifen (1uM); Homo sapiens; ChIP-Seq
Fixation was stopped with 2.5M glycine and cells were washed 2X in PBS/0.5% Igepal CA-630.  Cell pellets were snap frozen and chromatin 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. Genomic DNA regions of interest were isolated using antibodies against ERa (Santa Cruz polyclonal sc-543). 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 65ºC, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation.