Protocol for base resolution mapping of ac4C using RedaC:T-seq

Abstract

N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH₄-induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH₄ to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022).¹.

Keywords: Bioinformatics; Molecular Biology; RNAseq; Sequencing.

Graphical abstract

Figure 1

RNA integrity and efficiency of ribodepletion (A and B) Bioanalyzer profile of total RNA pre- and post-ribodepletion.

Figure 2

Reduction of ac4C NaBH₄ reduces ac4C to tetrahydro-ac4C (top). Reduced ac4C less efficiently base pairs with guanosine and can interact with adenosine instead (bottom).

Figure 3

RNA integrity after NaBH₄ treatment (A) Agarose gel electrophoresis of total RNA samples treated with NaBH₄ or left untreated. (B) Bioanalyzer profile of total RNA treated with 100 mM NaBH₄ for 1 h at 55°C.

Figure 4

DNA library integrity Bioanalyzer profiles of DNA libraries from HeLa WT and NAT10^−/− cells.

Figure 5

Sanger sequencing of 18S rRNA helix 45 after NaBH₄ treatment

Figure 6

Mismatches observed in RedaC:T-seq data (A) Bar plot of frequency of each mismatch type. Sample data from chromosome 19, sites selected at p < 0.01 and > 5 fold mismatch rate difference. (B) Browser view of a site in the SIPA1L3 gene. NaBH₄ treated samples in WT and NAT10^−/− are shown, along with untreated control.