GSM9464924: PT22_H3K4me3; Mus musculus; ChIP-Seq
Cells were crosslinked for 10 min with 1% w/v formaldehyde, quenched in 0.125 M glycine, washed twice with ice-cold PBS, scraped, and harvested by centrifugation. Cell pellets were sent on dry ice to Active Motif, Inc. (Carslbad, CA) for ChIP-Seq analyses as fee-for-service. Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp with Active Motif's EpiShear probe sonicator (cat# 53051). Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by SPRI beads clean up (Beckman Coulter) and quantitation by Clariostar (BMG Labtech). Extrapolation to the original chromatin volume allowed determination 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 4 µg of antibody against PAX8 (Proteintech 10336-1-AP) or specific histone modifications (H3K4me3-Active Motif 39159; H3K27me3-Active Motif 39155). 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 65⁰C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end).