GSM2265610: RCC4_SeqCapEpi_BSseq_r1; Homo sapiens; 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. Fragments were selected from these libraries using the SeqCap Epi CpGiant Enrichment Kit (Roche) following the manufacturers instructions, sequenced from both ends for 100 bases on a HiSeq 2000.