GSM4221295: WT 1 Input; Mus musculus; ChIP-Seq
Isolated myocytes were resuspended in PBS immediately after the last step of calcium reintroduction. Formaldehyde at 1% concentration was added onto the tube to cross-link the cells and tube was placed on a rocker at room temperature for 5 minutes. Then, glycine at 125mM concentration was added to the tube, which was rocked on ice for 5 more minutes. Cells were then washed 3 times in PBS and then lysed using a dounce on ice in a cell lysis buffer (5mM Na-butyrate, 10mM HEPES pH8.0, 85mM KCl, 0.6% NP-40, protease and phosphatase inhibitors). The lysates were centrifugated for 5 minutes at 2,000 rpm at 4ºC, resuspended in a cell lysis buffer and incubated on ice for 10 minutes. The percentage of free nuclei was assessed visually, the suspension was centrifugated, and resuspended in nuclear lysis buffer (50mM TrisHCl pH8.0, 10mM EDTA, 1% Triton X-100, 0.8% SDS, protease and phosphatase inhibitors). It was then homogenized and incubated for 10 minutes on ice with regular flicking, before being transferred to sonication tubes. The sonication was performed on a Bioruptor Pico for 20 cycles 30sec ON/30sec OFF cycles at +4ºC. The sheared chromatin was then collected after centrifugation for 10 minutes at +4ºC at 13,000 rpm, diluted 10-fold with ChIP dilution buffer (16.7mM TrisHCl, 1.2mM EDTA, 140mN NaCl, 1.1% Triton X-100 protease and phosphatase inhibitors). A 10% input was collected before adding 6µg of an anti-BRD4 antibody to the remaining chromatin. After overnight immunoprecipitation, a mixture of sepharose A/B beads, pre-blocked with 1% BSA, was added onto the tube for 1 hours. Immunoprecipitates were then washed sequentially with a Low Salt buffer (0.1% SDS, 1%TritonX-100, 2mM EDTA, 20mM TrisHCl pH8.0, 150mM NaCl), High Salt buffer (0.1% SDS, 1%TritonX-100, 2mM EDTA, 20mM TrisHCl pH8.0, 500mM NaCl), LiCl buffer  (1% NP-40, 1% Na-Deoxycholate, 1mM EDTA, 10mM TrisHCl pH8.0, 250mM LiCl), and then twice in TE buffer (10mM TrisHCl pH 8.0, 1mM EDTA). Chromatin elution was collected in 1% SDS, 10mM EDTA, 50mM TrisHCl pH 8.0 at +65ºC for 15 minutes with agitation. The supernatant was then agitated at +65 ºC overnight at room temperature with 200mM final of NaCl. Chromatin was treated with RNAse A for 2 hours at +37ºC, proteinase K for 1 hour at +55ºC and DNA was then extracted using Phenol/chloroform process followed by ethanol precipitation. Kapa Hyper Kit
GEO
GSM4221295
GEO Accession
GSM4221295