title: RNA Pol II-pS5 ChIP-Seq analysis of Wt mouse BMDM cells differentiated in the presence of MCSF (24hIL4_1hLPS_rep1)
design: Macrophages were double cross-linked with DSG (30min) and formaldehyde (10min). 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. Chromtain was sheared with sonication and immunoprecipitated overnight using IgG (Millipore, 12-370) and antibodies against RXR (sc-774), P300 (sc-585), PU.1 (sc-352), RAD21 (ab992), STAT6 (sc-981) and PPARG (Persseuss #PP-A3409A). Chromatin-antibody complexes are 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 5ng DNA either with Ovation Ultralow Library Systems (Nugen) or TruSeq ChIP library systems (Illumina) according to manufacturer_s instructions. Libraries were sequenced on HiSeq 2500.