GSM8386348: CTRL_Input; Mus musculus; ChIP-Seq
ChIP was performed on quadriceps from control and tumor-bearing mice, as previously described in Segatto et al., 2017 (PMID:29167426) and Savic et al., 2013 (PMID: 24279905). In brief, frozen tissues were pulverized using Covaris tissue TUBEs (Covaris 520001) with a chilled hammer on a metallic block placed in dry ice. The resulting powdered tissue was transferred to a glass vial and resuspended in 10ml 1xPBS, followed by cross-linking in 1% formaldehyde (Ted Pella Inc) for 15 minutes at RT. After blocking the cross-link with 0.125 M glycine for 5 minutes and washing with PBS containing protease and phosphatase inhibitors, the pellets were resuspended in Farnham buffer (5 mM PIPES pH 8.0; 85 mM KCl; 0.5% NP-40) and briefly homogenized with a homogenizer. Subsequently, cells were lysed in RIPA buffer (1× PBS; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS). Chromatin was sonicated to achieve fragment lengths of approximately 0.5 kb and then subjected to immunoprecipitation using 5 μg of CREB or phospho-CREB1 antibodies and Protein G Dynabeads (Thermofisher). Washes were performed as described in Segatto et al., 2017 (PMID:29167426). The eluted DNA was reverse-crosslinked, digested with 20 μg of Proteinase K for 1hr at 55C and purified using Qiagen MinElute PCR purification kit. Paired-end sequencing was performed on the Illumina NextSeq 1000 platform.