title: GSM9161480: mESC, Suz12-mAID-mClover, Control 1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were dissociated with 0.05% trypsin, fixed with 1% formaldehyde solution for 10 min at room temperature (RT), and quenched with fresh 125 mM glycine for 5 min RT. After PBS washed, nuclei were isolated using sequential lysis buffers: B1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x protease inhibi-tors; 10 min at 4°C), B2 (10 mM Tris-HCL, pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 1x pro-tease inhibitors; 10 min at 4°C), and B3 (10 mM Tris-HCL, pH 8, at 4 C, 100 mM NaCl,1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-Lauroylsarcosine sodium salt, and 1x protease inhibitors). Chromatin was fragmented to an average size of 250-500 bp using a Diagenode Bioruptor and triton X-100 was added to a final concentration of 1%. ChIP was performed using 1 ug of antibody per 2 million of cells incubated and rotated overnight at 4 ºC. Antibodies: rabbit anti-GFP (Abcam, ab290), rabbit anti-H3K27me3 (Cell signaling, 9733). Dynabeads (10 µL pero µg Ab) were pre-washed in PBS + 0.5% BSA and rotated for 2 hours at 4 ºC before adding to the IPs.  Then washes X5 with ChIP RIPA buffer 4°C (50mM HEPES-KOH, pH 7.5 at 4 °C, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na-deoxycholate) and one wash with TE + 50 mM NaCl. DNA was eluted in freshly prepared elution buffer (50 mM Tris, pH 8, 10 mM EDTA, 1% SDS) at 65 °C for 20 min and then de-crosslinked overnight at 65 °C. Samples were treated with RNase A and proteinase K and DNA was purified by phenol:chloroform:isoamyl alcohol (P:C:IA). Libraries were pre-pared using NEBNext Ultra II DNA Library Prep Kit (NEB), quantified by Qubit dsDNA HS Assay, quality-checked with Aligent Bioanalyzer HS DNA chip. Libraries were sequenced as 150 pb paired-end reads on the Illumina HiSeq platform.