GSM6213902: ChIP_primary_hepatocytes_VC; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde at room temperature for 15 min and quenched with 0.125 M glycine and shipped to Active Motif (Carlsbad, CA, U.S.A) for ChIP-seq analysis. Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated using a Misonix Sonicator 3000. For each ChIP reaction, 30 μg of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reactions were set up using precleared chromatin and a rabbit polyclonal antibody against AhR (ENZO Lifesciences, BML-SA210, Lot# 01031447) and incubated overnight at 4 °C. Protein A agarose beads were added and incubation at 4 °C was continued for another 3 h. Immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. Immune complexes were eluted from the beads with SDS buffer and subjected to RNase treatment 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. Genomic DNA was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA was quantified on a NanoDrop spectrophotometer. .equipped with a microtip in order to shear the DNA to an average length of 300–500 bp. Lysates were cleared by centrifugation and stored at −80 °C.  ChIP and Input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3′-ends. Illumina genomic adapters were ligated and the sample was size-fractionated (200–250 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles), the resulting DNA libraries were quantified and sequenced on HiSeq 2000.