Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs

Detection of ac4C in 18S rRNA through RetraC:T and nanopore sequencing. (A) Agarose gel analysis of total RNA integrity after treatment with NaCNBH₃ for 5 min. (B) Schematic of the nanopore-PCR sequencing strategy to evaluate the impact of 2-amino-dATP on 18S rRNA sequencing. (C) Browser views detailing 18S rRNA read coverage in sequencing results from untreated (HCl) and NaCNBH₃-treated RNA from wild-type and NAT10^−/− HeLa cells reverse transcribed with dNTPs alone or supplemented with 2-amino-dATP. At an allele threshold of 0.15, the ac4C site at nt 1842 is the only readily visible mismatch due to sequencing coverage drop-off. (D) Browser views surrounding the ac4C sites at 18S nt 1337 and 1842 from nanopore sequencing of untreated or NaCNBH₃-treated wild-type and NAT10^−/− HeLa RNA reverse transcribed in the presence of 2-amino-dATP or dNTPs alone, as shown. C:T mismatches are depicted in red. (E) Cumulative mismatch rates from nanopore sequencing of untreated or NaCNBH₃-treated wild-type and NAT10^−/− HeLa 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. (F) Browser views surrounding m1acp3Ψ at 18S nt 1248 from nanopore sequencing of untreated or NaCNBH₃-treated RNA reverse transcribed in the presence of 2-amino-dATP or dNTPs alone, as shown. T:C mismatches are depicted in blue.