GSM4600752: HEK293T NSUN6 KO; Homo sapiens; Bisulfite-Seq
Total RNA was isolated with TRIzol reagent and Direct-zol RNA MiniPrep kit; for cytoplasm/nuclear fractions, cells were fractionated using the PARIS kit (Ambion) according to the manufacturer's instructions. Polyadenylated RNA was separated from total RNA using Oligo dT Magnetic Beads (Vazyme). 100ng-1μg of polyadenylated RNA was converted using the EZ RNA methylation kit (Zymo Research) with 3 cycles of [70 C 10 min, 64 C 45 min] and 37 C 30 min for desulfonation. The converted RNA was fragmented into 150~200 nt fragments by incubation at 94 °C for 8 minutes in fragmentation buffer (NEBNext Ultra II Directional RNA Library Prep Kit, NEB). The fragmented RNA was then used for library construction, following the manufacturer's protocol (NEBNext Ultra II Directional RNA Library Prep Kit, NEB).