GSM6214413: ChIP-seq_D542G_ER_rep1; Mus musculus; ChIP-Seq
Minced mammary glands were fixed in formalin, then used for chromatin preparation. Chromatin was isolated by disruption with a Dounce homogenizer in lysis buffer. 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. 30 µg of chromatin was precleared with protein A agarose beads (Invitrogen), then genomic DNA regions of interest were isolated using 4 µg of antibody against ERα (Santa Cruz, sc-543) or PR (Santa Cruz, 7208). 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. IIlumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation.