GSM8999216: DNMT1 KO WGBS; Homo sapiens; Bisulfite-Seq
DNA was extracted from cell pellets snap-frozen in dry ice with ethanol. Cells pellets were resuspended in Genomic Lysis Buffer (300mM NaCl, 1% SDS, 20mM EDTA). The lysis mixture was syringed up and down using a 21G needle before being incubated at 55°C overnight with proteinase K. RNA was removed by incubation with RNase A/T1 Cocktail (Ambion) at 37 °C for 1 h, in between two phenol–chloroform extraction steps. DNA was quantified by Nanodrop 8000 spectrophotometer or Qubit fluorometer (Invitrogen) and purity was assessed using the Agilent 2100 BioAnalyzer. DNA (100ng) was bisulphite converted using the EZ DNA Methylation-Lightning kit, according to the manufacturer's instructions. Libraries were generated using the TruSeq DNA Methylation kit, according to the manufacturer's instructions. Sequencing was performed at the Edinburgh Clinical Research Facility. Libraries were sequenced using the Illumina NextSeq 500/550 High-Output v2 (150 cycles) (#FC-404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002) over 1 flow cell.