Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

5-Methylcytosine (m⁵C) is one of the posttranscriptional modifications in mRNA and is involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m⁵C mapping still needs improvement. Here, we develop a detection method named DRAM (deaminase and reader protein assisted RNA methylation analysis), in which deaminases (APOBEC1 and TadA-8e) are fused with m⁵C reader proteins (ALYREF and YBX1) to identify the m⁵C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provides transcriptome-wide editing regions which are highly overlapped with the publicly available bisulfite-sequencing (BS-seq) datasets and allows for a more stable and comprehensive identification of the m⁵C loci. In addition, DRAM system even supports ultralow input RNA (10 ng). We anticipate that the DRAM system could pave the way for uncovering further biological functions of m⁵C modifications.