GSM2265617: mES Tet-TKO_WGBS 4; Mus musculus; Bisulfite-Seq
cultured cells were washed on ice with ice-cold phosphate-buffer saline (PBS), detached using cell scrapers and collected by centrifugation (400 ×G, 4°C). Nucleic acids were subsequently extracted using the Wizard Genomic DNA Purification (Promega, Leiden, The Netherlands) kit according to instructions, dissolved in 80 µL PBS with RNAse A (200 units, NEB, Ipswich, MA, USA), incubated for 10 minutes at 37°C. After proteinase K addition (200 units) and incubation for 30 minutes at 56°C, DNA was purified using the QIAQuick blood and tissue kit, eluted in 100 µL of a 10 mM Tris, 1mM EDTA solution (pH 8) and stored at -80°C until further processing. DNA libraries were prepared using methylated adapters and the NEBNext DNA library prep master mix set following manufacturer recommendations. Libraries were bisulfite-converted using the Imprint DNA modification kit (Sigma) as recommended, and PCR amplified for 12 cycles using barcoded primers (NEB) and the KAPA HiFi HS Uracil+ ready mix (Sopachem, Eke, Belgium) according to manufacturers instructions.