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

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.