Bisulfite-sequencing analysis reveals no m5C sites in SARS-CoV-2 RNA. (A) Schematic representation of SARS-CoV-2 genome structure. A 316 nt sequence stretch of the N protein-encoding region harboring eight putative m5C sites (Taiaroa et al. 2020) is highlighted, and the m5C candidate sites are marked in red (numbers corresponding to NC_045512v2 sequence). (B,C) Bisulfite-treated RNA from virus-infected Vero E6-TMPRSS2-ACE2 (B) or human Caco-2 cells (C) was reverse transcribed, and the region indicated in (A) was amplified by PCR followed by subcloning and Sanger sequencing of individual clones (n = 35 for each cell line). No unconverted Cs were detected at the indicated positions in viral RNA. Methylated positions C38, C47, and C48 in human tRNAAsp are shown as positive control (n = 20). (D) Read coverage of SARS-CoV-2 RNA in three biological replicates after RNA-seq of bisulfite-treated RNA from infected Vero E6-TMPRSS2-ACE2 cells.
