m1A reduction with NaBH4 improves mutation rate and readthrough rate in RT. (A) A gel image of readthrough assay of 33-mer RNA oligonucleotide containing m1A without treatment (control, C) or treated with NaBH4 (R) using different RT enzymes. (P) Primer, (T) truncated, (FL) full length, (C) control, (R) reduction (top). A plot representing quantified readthrough/truncation ratios in readthrough assay. (Ctrl) Control, (Red) reduction (bottom). (B) Sanger sequencing chromatograms of 33-mer oligonucleotide containing m1A. RNA was untreated (control) or treated with NaBH4 followed by RT with different RT enzymes followed by PCR and Sanger sequencing. (C) Fluorescence intensity measured in the RT-PCR-IVT assay using different RT enzymes with the synthetic 33-mer oligonucleotide containing U, A, or m1A at position 15 with or without reduction treatment as the substrate. (D) Bar plot showing the mutation rate (%) at the m1A modified site in the spike-in RNA oligo with a fixed sequence. These libraries were constructed using the NaBH4-, pNTP-, or Tris-HCl (pH 8.8)-treated and SSIV and PSII RT enzymes. (E) Pie charts showing the mutation patterns of m1A-modified sites in the context of “AAm1AGC” within the spike-in RNA oligo as observed in PSII (top) and SSIV (bottom) RT libraries. (F) The heatmap showing the mutation rates at the m1A site in the NNm1ANN spike-in oligos in the reduction libraries constructed using the PSII RT enzyme. The mutation rates were determined by NGS. Mutation rates in percentage are color-coded. (G) Snapshots of IGV coverage tracks showing the mutation signatures of m1A1,322 site in human 28S rRNA under different library construction conditions.
