GSM1973390: input; Mus musculus; ChIP-Seq
Tumor nodules or control liver tissue were washed in PBS, cut into pieces ≤ 2mm diameter and fixed for 15 min in 1% of formaldehyde (Sigma) in PBS. Tissue was washed in PBS and stored at -80°C until further processing. Upon thawing, tissue was incubated in 125 mM glycin (5 min, room temperature), washed in PBS and nuclei were prepared on ice using a dounce homogenizer. All following steps were performed on ice, if not specified otherwise. 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, 0.2% 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 once with proteinA-sepharose and once with proteinG-sepharose beads, both blocked with 0.5% E. coli tRNA (Sigma) and 0.5% BSA. In this study, the following antibodies were used: Myc N262 (Santa Cruz, sc-764X), RNA Pol II N20 (Santa Cruz, sc-899X), H3K4me1 (Abcam, ab8895), H3K4me3 (Active Motif, 39159), and H3K27ac (Abcam, ab4729). Normal rabbit IgG (Santa Cruz, sc-2027) was used as background control.  For Myc and RNA Pol II, 10 µg of antibodies were incubated overnight with 500µl of chromatin (+ 500µl of ChIP buffer to each a volume of 1ml per reaction). Blocked protein G-sepharose beads were added, incubated for 2-3h 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, 0.2% 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, 0.2% 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, 0.2% NaN3) and once with TE. DNA was de-crosslinked overnight (2% SDS in TE, 65°C), purified using QiaQuick columns (Qiagen) and quantified using PicoGreen (Invitrogen). For histone marks, 5 µg of antibody were pre-bound overnight to proteinG-coupled paramagnetic beads (Dynabeads, Invitrogen) in PBS/BSA 0.5%. Beads were washed and added to the chromatin (50-100 µl in a total volume of 1ml of ChIP buffer) and after overnight incubation washed 7x in RIPA-like buffer (50mM HEPES pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% sodium deoxycholate) and once in TE containing 50mM NaCl. DNA was eluted from the beads in TE-2% SDS (15 min, 65°C), de-crosslinked overnight (65°C), purified and quantified using PicoGreen (Invitrogen). Blecher-Gonen R1, Barnett-Itzhaki Z, Jaitin D, Amann-Zalcenstein D, Lara-Astiaso D, Amit I., High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states. Nat Protoc. 2013 Mar;8(3):539-54