GSM2790894: anti_Brd4_cachexia; Mus musculus; ChIP-Seq
Frozen tissues were pulverized in Covaris tissue TUBEs (Covaris 520001) using a chilled hammer on a cold metallic block, in dry ice. Powdered tissue was resuspended in PBS, cross-linked in 1% formaldehyde for 15 minutes. After blocking crosslink with 0.125M glycine for 5 minutes and washes with PBS plus protease and phosphatase inhibitors, pellet was resuspended in Farnham buffer (5 mM PIPES pH 8.0; 85 mM KCl; 0.5% NP-40) and briefly homogenized with a tissue homogenizer.   Cells were subsequently lysed in RIPA buffer (1x PBS; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS). 100mg of starting tissue was used for each antibody. Chromatin was sonicated to fragments length of approximately 0.5Kb and immunoprecipitated with 3.5µg of BRD4 antibody. Immunoprecipitation procedures were performed as in Proserpio et al., 2013. For ChIPSeq,  10ng of immuno-precipitated DNA fragments were used to prepare ChIP-seq libraries with  Ovation SP Ultralow DR Multiplex system (Nugen) following the manufacturer’s protocol.