GSM8443992: Input, ESCs; Mus musculus; ChIP-Seq
ESCs were cultured under optimum conditions and were fixed at a high‐confluence stage at room temperature for 15 min using 1% formaldehyde in fixing buffer, followed by quenching with 0.125 M glycine at room temperature for 7 min. Cells were resuspended in lysis buffer [50 mM Hepes (pH 7.9), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP‐40, 0.25% Triton X‐100] and then in sonication buffer [0.1% SDS, 1 mM EDTA, 10 mM Tris (pH 8.1)]. Chromatin shearing was carried out in the Covaris S2 sonicator using the Covaris TC12 × 12 mm tubes (Tube AFA Fiber and Cap, Covaris) for 12 min (duty factor: 75, peak power: 25, cycles per burst: 200) allowing the shearing of chromatin within a range of 250–500 base pairs DNA fragments. Triton X‐100 and NaCl were then added in the sheared chromatin to final concentrations of 1% and 150 mM, respectively. ChIPs were carried out by incubating 100 μg of chromatin with the proper dilution of the anti-Nanog antibody (1:100) per ChIP reaction [anti‐Nanog, D1G10 Rabbit mAb ChIP Formulated #8785, Cell Signalling]. Next, protein G‐Dynabeads (10004D, Thermo Fisher Scientific) pre‐equilibrated in IP buffer [0.1% SDS, 1 mM EDTA, 10 mM Tris (pH 8.1), 1% Triton X‐100, 150 mM NaCl] were incubated with the chromatin‐antibody solution in an orbital mixer at 4°C for 4 hours. The recovered resin was subsequently washed with 150mM NaCl buffer (1%Triton, 0.1%SDS, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl), 250 mM  NaCl buffer (1%Triton, 0.1%SDS, 2mM EDTA, 20mM Tris pH 8.0, 250mM NaCl), once with LiCl buffer (0.25M LiCl, 1% NP-40, 1% DOC, 1mM EDTA, 10mM Tris pH 8.0) and once with TE (1mM EDTA, 10mM Tris pH 8.0). The captured chromatin fragments were subjected to 30ug RnaseA for 1 hr at 37°C , then 40ug/ml PK for 1 hr at 50°C  and finally 0.3M NaCl at 65°C overnight for reverse crosslinking. All DNA present in each sample was purified with Nucleomag DNA purification beads (Macherey-Nagel) and eluted in TE buffer. Libraries were constructed using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, according to the manufacturer's guidelines.