alias: E-MTAB-7593:liver_71_1B_s
title: Illumina HiSeq 2000 sequencing; ChIP-sequencing analysis of RNA Pol II binding sites 4 hours after Myc activation in liver and heart tissue, from wild type and R26CAG-c-MycERT2 mice
design: ChIP-sequencing analysis of RNA Pol II binding sites 4 hours after Myc activation in liver and heart tissue, from wild type and R26CAG-c-MycERT2 mice
library_construction_protocol: Adult animals requiring 4-OHT were either i.p. injected with 1 mg (Z)-4-hydroxytamoxifen (4-OHT; Sigma, H7904) in 10 % ethanol and vegetable oil (5 mg/ml) and collected 4 hours post injection. Mouse tissue was harvested from mice, washed in PBS and cut into small 2-3mm pieces with a scalpel blade. Tissue was then fixed in 1% formaldehyde/PBS for 15 min, washed in PBS 3 times, snap frozen and stored at -80°C until further processing. Upon thawing, tissue was incubated in 125 mM glycine (5 min, room temperature), washed in PBS and nuclei prepared on ice using a dounce homogenizer. All following steps were performed on ice. Nuclei were washed in PBS, resuspended in ChIP buffer (4 ml of buffer per 0.5 ml of tissue; 100mM NaCl, 133mM Tris pH 8.0, 5 mM EDTA, 0.02% NaN3, 0.67% SDS, 1.67% Triton X-100) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche) and phosphatase inhibitors (50 mM NaF, 1 mM β-GP, 1 mM NaV). Chromatin was sonicated to an average length of 300 bp, pre-cleared twice with protein G-sepharose beads, pre-blocked with 0.5% E. coli tRNA (Sigma) and 0.5% BSA. In this study, the following antibody was used: Rabbit Polyclonal anti-RNA Pol II N20, Santa Cruz Cat# sc-899X. 10 µg of antibodies were incubated overnight with 500 µl of chromatin (+ 500µl of ChIP buffer to each a volume of 1ml per reaction). Blocked protein G-sepharose beads were added, incubated for 2-3 hours and washed as follows: 3X with mixed micelle buffer (150 mM NaCl, 20 mM Tris pH 8.1, 5 mM EDTA, pH 8.0, 5.2% sucrose, 0.02% NaN3, 1% Triton X-100, 0.02% SDS), 2x with buffer 500 (50 mM HEPES pH 7.5, 0.1% sodium deoxycholate, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.02% NaN3), 2x with LiCl/detergent buffer (10 mM Tris pH 8.0, 0.5% sodium deoxycholate, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.02% NaN3) and once with TE. DNA was de-crosslinked overnight (2% SDS in TE, 65°C), purified using QiaQuick columns (Qiagen) and quantified using Qbit (ThermoFisher).  2-5 ng of ChIP DNA were used for ChIP-seq library preparation.
identifiers.namespace: UNIVERSITY OF CAMBRIDGE
identifiers.id: E-MTAB-7593:liver_71_1B_s
attributes.tag: Experimental Factor: organism part
attributes.value: liver
attributes.tag: Experimental Factor: genotype
attributes.value: Rosa26-CAG-MycER/+
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-Pol II