Figure 2. Sequencing by chemical modification for the location of modified bases.

a, Chemical treatments are applied to the modified bases pseudouracil and hypoxanthine, and the structures that result prevent the read through of RNA transcripts by reverse transcriptases. b, Sequencing by modification-specific termination of reverse transcription. The process for pseudouridine is shown as an example^8–10. Poly(A)⁺-enriched mRNA is fragmented and treated with CMC. A 3′-adaptor is ligated to each fragment and then reverse transcription is carried out. Next, truncated complementary DNA is selected by gel electrophoresis, amplified and sequenced. The location of the modification site is determined by comparing the frequency of read termination with and without the CMC treatment. c, The effect of sodium bisulfite (NaHSO₃) treatment on m⁵C and cytosine. The resulting structures are shown on the right. d, Bisulfite sequencing²⁵. Poly(A)⁺-enriched mRNA is fragmented and treated with sodium bisulfite. In the treated sample, all cytosines are converted to uracils; however, m⁵C is resistant to the treatment. The location of this base modification can be then identified as the sites that still code as cytosine after sodium bisulfite treatment. C, cytosine; U, uracil.