Investigating the effect of ALKBH5 on HBV replication by Long Read sequencing of the viral transcriptome with and without an m6 methylation site in the basic core promoter under normoxic (18%) or hypoxic (1%) oxygen.
150 ng of RNA prepared from normoxic or hypoxic HepG2-NTCP cells transfected with either WT or m6-null (m1) HBV1.3 was reverse transcribed with barcoded sequencing primers. HBV cDNAs were hybridised to biotinylated 120 bp long oligos targeting the entire HBV genome (designed using the IDT xGen Lockdown platform) and enriched with an xGen Hybridization kit (IDT). The captured DNA sequences were pooled and SMRTbell library ligation, amplification and sequencing performed in accordance with the manufacturers instructions (PacBio).