GSM3045304: Terminal Effector H3K27ac Kaech 1; Mus musculus; ChIP-Seq
ChIP experiments were performed on FACS purified in vivo adoptively transferred P14+ CD8+ T cells. CD8α+CD44+Thy1.1+ cells were sorted based on expression of KLRG1 and IL7Rα to purified populations of TE (KLRG1HiIL7RαLo) and MP (KLRG1LoIL7RαHi) cells. Cells were crosslinked with 1% formaldehyde in 10% fetal bovine serum containing RPMI medium for 10 minutes at 37C. Crosslinking was stopped by addition of 2.5M glycine at 1:20 dilution for 5 minutes at room temperature. Washed cells were lysed and sonicated to obtain chromatin fragments of 150 to 500 base pairs. ChIP was performed on sonicated chromatin from 1-10 million cells with anti- H3K27me3 (Abcam, ab6002), anti-H3K27Ac (Abcam, ab4729), anti-H3K4me3 (Abcam, ab4XXX), and anti-H3K4me1 (Abcam, ab4XXX) antibodies. ATAC-seq library preparations were performed as described in (Buenrostro et al., 2013). Immunoprecipitated DNA was purified, amplified, processed into a library, and sequenced on an Illumina HiSeq 2500 with 4 samples per lane (170M reads are distributed at 42.5M reads per sample with 75bp reads in single-end mode). Total RNA was purified with the use of a Qiazol and RNeasy Mini kit (Qiagen), in which on-column treatment with DNase was included. Purified RNA was submitted to the Yale Center for Genomic Analysis, where it was subjected to isolation of mRNA and library preparation. Libraries were pooled, six samples per lane, and were sequenced on an Illumina HISEQ 2500 (75–base pair paired- end reads), followed by alignment and quantification with Kallisto using the GRCm38 (mm10) reference genome. Counts were imported and normalized with DESeq2 v1.14.1, with the read counts normalized by fitting the data to a local smoothed dispersion in an unblinded fit to better capture the observed dispersion-mean relationship and account for batch effects, and subsequently used to perform differential expression analysis (Love et al., 2014).
GEO Accession
GSM3045304