Fig. 2 |. Experimental workflow for ac4C-seq protocol.

RNA is spiked with a synthetic ac4C RNA spike-in and split into three samples. One experimental sample is treated with NaCNBH₃ under acidic conditions (reduction, positive signal), while two control samples are subjected to acidic conditions without reducing agent (mock-treated, control no. 1) and deacetylation followed by NaCNBH₃ treatment (deacetylated + reduction, control no. 2). A 3′ adapter is ligated onto fragmented RNA, which is then reverse transcribed, resulting in misincorporation of ‘A’ in cDNA at positions of reduced ac4C. A 3′ adaptor is ligated to facilitate cDNA library construction, with subsequent sequencing and bioinformatic analysis being used to identify ac4C-modified sites. Figure adapted with permission from ref. ⁵. Red., reduced.