title: PPARg ChIP-Seq analysis of mouse E14 embryonic stem cells differentiated to preadipocytes (day 21, rep1)
design: E14 mouse embryonic stem cells were differentiated to preadipocytes as described (Cuaranta-Monroy et al. Stem Cell Res. 2014). Preadipocytes at day 21 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. Chromatin was sheared with sonication and immunoprecipitated overnight using antibodies against RXR (sc-774) and PPARG (Persseuss #PP-A3409A). 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 5ng DNA either with Ovation Ultralow Library Systems (Nugen) or TruSeq ChIP library systems (Illumina) according to manufacturers instructions. Libraries were sequenced on HiSeq 2500.