GSM7429952: OT-I T cells expanded with IL2 and stimulated with OVA peptide, vehicle treated control, H3K27me3 pulldown, replicate2; Mus musculus; ChIP-Seq
In brief, cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. 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 (40 ug) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ug of antibody against H3K27me3 (Active Motif, 39155, Lot 11822023). 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. Illumina sequencing libraries (a custom type, using the same paired read adapter oligonucleotides described by Bentley et al., (2008)) were prepared from the ChIP and Input DNAs on an automated system (Apollo 342, Wafergen Biosystems/Takara).