RNA Bisulfite Sequencing Protocol

We performed RNA bisulfite treatment (bsRNA-seq) following the protocol from Johnson et al.⁶⁴ . There were no deviations done for the protocol except the fact that a GE50 spin column was used for the removal of the excess of bisulfite reagent (sodium bisulfide and hydroquinone) instead of a GE25. This protocol was applied to RNA fully modified with m5C or non-modified obtained from in-vitro transcripts (IVTs) m1, m2, m3, m4 (Suppl. Data S9) built from four non-overlapping fragments from the mouse canonical pre-rRNA (~13 kb long). Sequencing of the bisulfite-treated samples was performed with Illumina. For the analysis of the Illumina reads from the bisulfite-treated data we used meRanTK^{65 (link)}, adjusting the parameters to make it more permissive to m5C detection: the edit distance was changed from the default 2 – 200, the number of Cs per Illumina read was changed from the default 3–200, the minimum methylation ratio of a single C needed for methylation was changed from the default 0.2–0, and the minimum coverage at a given reference site above which methylation call is performed was changed from the default 20–0. The same modified and non-modified RNA samples were used to perform nanopore DRS.