PU.1 ChIP-seq analysis of human differentiating macrophages treated with IL-4 (rep2)
Human CD14+ monocyte-derived macrophage-like cells 24 hours following isolation were treated with IL-4 (20 ng/ml, 30 min) then harvested by scraping in medium followed by centrifugation. Cells were double cross-linked with DSG (30 min) and formaldehyde (10 min). Nuclei were isolated using lysis buffer containing 0.15M NaCl, 0.005M EDTA pH 7.5, 0.05M Tris-HCl pH 7.5, 0.5% NP40, supplemented with protease inhibitor (Roche) prior to use. Chromatin was sheared with sonication and immunoprecipitated overnight using antibodies against RXR (sc-774), PU.1 (sc-352), STAT6 (sc-981) and H3K27ac (ab4729). Chromatin-antibody complexes were extensively washed and eluted after pull-down by paramagnetic beads. Eluted complexes were decrosslinked overnight and purified by Minelute PCR Purification Kit. Immunoprecipitated DNA was quantified by Qubit fluorometer. Libraries were prepared from 5 ng DNA either with Ovation Ultralow Library Systems (Nugen) or TruSeq ChIP library systems (Illumina) according to the manufacturerŐs instructions.