Protocol for profiling RNA m⁵C methylation at base resolution using m⁵C-TAC-seq

RNA 5-methylcytosine (m⁵C) is a widespread modification and plays a crucial role in gene expression regulation. Here, we present a protocol for transcriptome-wide m⁵C methylome profiling at base resolution using bisulfite-free m⁵C detection strategy enabled by ten-eleven translocation (TET)-assisted chemical labeling sequencing (m⁵C-TAC-seq). We detail steps for TET-assisted chemical labeling, library construction, and data analysis. m⁵C-TAC-seq enables accurate and robust m⁵C detection in various RNA species. For complete details on the use and execution of this protocol, please refer to Lu et al.¹.