GSM6032224: H3K27ac, Mac, GRLysMCre, replicate3; Mus musculus; ChIP-Seq
20 million bone marrow derived macrophages were changed to Macrophage serum free medium (Gibco, 12065-0723) overnight before treatment with IL-4 (20ng/ml) and Dexamethasone (100nM) for 2 hours. Cells were washed twice with ice cold PBS then fixed for 15 minutes with 1% formaldehyde (ThermoScientific 28908) followed by quenching with 1M Glycine (Sigma, 33226) for 5 minutes at room temperature. After washing twice with ice cold PBS cells were homogenized with a Douncer (Active Motif, 40415) in  Fast IP buffer (150mM NaCl, 50mM Tris-HCl (pH7.5), 5mM EDTA, 0.5%v/v NP-40, 1%v/v Triton X-100) with protease inhibitors (Complete Tablets EDTA-free, EASYpack, Roche, 04693132001). Resulting nuclei were centrifuged at 12,000 RPM and resuspended in shearing buffer (1% SDS, 10mM EDTA pH 8, 50mM Tris pH8) and sheared in 1.5ml Bioruptor Microtubes (Diagenode, C30010016) in a Bioruptor (Diagenode). Resulting sheared chromatin was cleared by centrifugation for 10 minutes at 12,000 RPM, 4C before diluting 1/10 ChIP-seq libraries were constructed from 10 to 20 ng of genomic DNA according to the manufacturer's instructions (Illumina) as described in (Nielsen R, et al. 2014. Methods in Enzymology 537: 261-279).