Linear and specific detection of ac4C through RetraC:T and Illumina sequencing. (A) Browser views detailing 18S rRNA read coverage in Illumina sequencing results from untreated and NaBH4-treated RNA reverse transcribed with HIV and SSIV RTase with dNTPs alone or supplemented with 2-amino-dATP. Visible mismatches at a 0.15 allele threshold correspond to known modifications, as shown. (B) Plot showing percent C:T mismatches at 18S nt 1337 versus percent wild-type HeLa RNA in a mixture with NAT10−/− RNA in RetraC:T-Illumina-seq with HIV and SSIV RTase. The gray dotted arrow indicates the C:T mismatch level in parallel processed samples from 100% WT RNA reverse transcribed with HIV or SSIV RTase and dNTP alone. (C) Browser views surrounding the ac4C site at 18S nt 1337 from Illumina sequencing of untreated or NaBH4-treated RNA reverse transcribed in the presence of 2-amino-dATP or dNTPs alone, as shown. C:T mismatches are depicted in red. (D) Cumulative mismatch rates from sequencing of untreated or NaBH4-treated RNA reverse transcribed in the presence of 2-amino-dATP or dNTPs alone, derived from each of the 1869 nt in 18S rRNA and segregated by modification status. (E) Browser views surrounding m1acp3Ψ at 18S nt 1248 from Illumina sequencing of untreated or NaBH4-treated RNA reverse transcribed in the presence of 2-amino-dATP or dNTPs alone, as shown. T:C mismatches are depicted in blue.
