title: GSM8485702: ChIP, Nanog, SOX2OFF, MDON, rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Briefly, cells were collected after trypsinization and fixed with 2 mM DSG in PBS for 50 min at room temperature on a rotating platform. Subsequently, cells were pelleted, and fixed for 10 min with 1 % formaldehyde on a rotating platform. The reaction was stopped using 200 mM Tris-HCl pH 8.0 for 10 min on a rotating platform. Tubes were filled with 1 % FBS in PBS and cells were spun down. Nuclei were extracted by resuspending the cell pellet twice in LB1 (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100), incubated for 10 min at 4°C and with gentle shaking at 100 rpm, and pelleted. The nuclei were then resuspended in LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), incubated for 10 min at 4°C and with gentle shaking at 100 rpm, and spun down. To wash nuclei, SDS shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.15% SDS) was added twice without disturbing the pellet, the tube was rolled to rinse the walls and spun down. All buffers were supplemented with a 1:100 dilution of protease inhibitor cocktail. Chromatin was sheared for 20 min at 5% duty, 140 W, 200 cycles, or for samples with 50,000 cells for 10 min, 10% duty, 75 W, 200 cycles in a Covaris E220 focused ultrasonicator. To remove the insoluble material, chromatin was spun down and the supernatant was transferred to a new tube. As total input control and to evaluate the sonication efficiency, a few microliters of chromatin were incubated with 1X TE buffer pH 8.0 and 10 ng/µl RNAse A at 37°C for 30 min. Proteinase K was added at 400 ng/µl and the reaction was incubated for another 30 min at 55°C and shaking at 1100 rpm. Finally, 200 mM NaCl was added to reverse the crosslinks at 65°C for 16 h and shaking at 1100 rpm. The chromatin was purified using a MinElute PCR purification kit (Qiagen, 28004) and quality-checked on a 1 % agarose gel. Next steps were performed using the ChIP-IT High Sensitivity kit (Active motif, 53040) with a few modifications highlighted below. For immunoprecipitation of samples made from at least 1 million cells, 3.5 µg chromatin were used unless stated otherwise, and for samples made from 50,000 cells, all extracted chromatin was used. The following antibodies were used: anti-OCT4A (Cell Signaling Technology, 5677S) at 3.1 µl per 3.5 µg chromatin, anti-SOX2 (Cell Signaling Technology, 23064) at 3.1 µl per 3.5 µg chromatin, and anti-NANOG (Cell Signaling Technology, 8822) at 1.55 µl per 3.5 µg chromatin. To obtain quantitative ChIP-seq results, drosophila spike-in chromatin and spike-in antibody (Active motif, 61686) were used according to the manufacturer's recommendations. Before binding the immunoprecipitated chromatin to the beads, the beads were washed once with ChIP buffer. For cross-link reversal, 2 µl proteinase K was added, the reaction was incubated at 55°C for 30 min and the temperature increased to 65°C and shaking at 1100 rpm for 16 h. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645L), and sequenced on a NextSeq 500 using 75-nucleotide read length paired-end sequencing. Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (NEB, E7645L)