GSM5492855: p65 STAT6WT (WT) 1-h LPS treated BMDM ChIP-seq rep 1; Mus musculus; ChIP-Seq
For RNA-seq, wild type (C57/B6 or Egr2 +/+), Stat6 -/- and Egr2fl/fl macrophages were differentiated in the presence of M-CSF using L929 cell supernatants for 6 days. On the 6th day, cells were re-plated and treated with IL-4 (20ng/ml) for 24 hours or left untreated. Then, the IL-4-primed and non-polarized macrophages were activated by 3 hours LPS stimulation. Poly-A tailed RNA molecules were pulled down with poly-T oligo attached magnetic beads. Following purification, mRNA was fragmented then cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second strand synthesis was performed followed by end repair, single 'A' base addition and ligation of barcode-indexed adaptors to the DNA fragments. ChIP was performed essentially as previously described (Daniel et al., 2014b), (Daniel et al., 2014a). ATAC-seq was carried out as described earlier with minor modification (Buenrostro et al., 2013). For RNA-seq adapter-specific PCRs were performed to generate sequencing libraries. Approximately 2.5ug was used for library preparation with TruSeq RNA Sample Preparation Kit (Illumina). Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). Libraries were sequenced on HiSeq 2500 instrument.