Acetylated cytidines are inefficiently captured through available methods. (A) Schematic depicting altered base-pairing following ac4C reduction to tetrahydro-ac4C (H4-ac4C). (B) Agarose gel analysis of total RNA treated with NaCNBH3 for 20 min at 20°C or NaBH4 for 1 h at 55°C. These conditions were used in the ac4C-seq and RedaC:T-seq studies, respectively. (C) Agarose gel analysis of total RNA integrity after treatment with NaCNBH3 for the indicated times. (D) Sanger sequencing surrounding the ac4C site in 18S rRNA helix 34 with cDNA produced from the NaCNBH3 time course shown in C. RT was performed with TGIRT. (E) Mass spectrometric analysis of ac4C and C levels in an RNA oligo containing a single ac4C after reduction with NaBH4 or NaCNBH3. An unmodified RNA oligo with a single C serves as control. Data represent the Mean ± SD as a percent of detected guanosine, n = 3. (F) NaBH4 or NaCNBH3 reduction of total RNA and cDNA synthesis with the NEBNext or TGIRT enzymes, followed by PCR amplification with primers flanking ∼90%–100% acetylated sites in 18S rRNA helices 34 and 45. Sanger sequencing results surrounding the known ac4C sites as indicated. (G) Reduction of an RNA oligo containing a single C or ac4C as depicted and cDNA synthesis with the NEBNext or TGIRT enzymes, followed by primer extension assay with a radiolabeled primer.
