biostudies-literatureCarissimi CSapienza Università di RomaMIURSapienza University of Rome2226-2235https://www.ebi.ac.uk/biostudies/studies/S-EPMC8648262biostudies-literatureUnknown18(12)In the last decade, the field of epitranscriptomics highlighted a wide array of post-transcriptional modifications in human RNAs, including microRNAs (miRNAs). Recent reports showed that human miRNAs undergo cytosine methylation. We describe the first high-throughput NGS-based method (BS-miRNA-seq) and an analysis pipeline (MAmBA) to attain high-resolution mapping of (hydroxy)-methyl-5-cytosine ((h)m5C) modifications in human miRNAs. Our method uncovers that miRNAs undergo widespread cytosine modification in various sequence contexts.Furthermore, validation of our data with specific antibodies reveals both m5C and hm5C residues in human mature miRNAs. BS-miRNA-seq and MAmBA may contribute to the precise mapping of (h)m5C on miRNAs in various cell types and tissues, a key achievement towardRNA biologyBisulphite miRNA-seq reveals widespread CpG and non-CpG 5-(hydroxy)methyl-Cytosine in human microRNAs.PMC8648262RM11816429252FCFFulci VDe Paolis VLorefice EAzzalin GLaudadio ICarissimi CfalseBisulphite miRNA-seq reveals widespread CpG and non-CpG 5-(hydroxy)methyl-Cytosine in human microRNAs.In the last decade, the field of epitranscriptomics highlighted a wide array of post-transcriptional modifications in human RNAs, including microRNAs (miRNAs). Recent reports showed that human miRNAs undergo cytosine methylation. We describe the first high-throughput NGS-based method (BS-miRNA-seq) and an analysis pipeline (MAmBA) to attain high-resolution mapping of (hydroxy)-methyl-5-cytosine ((h)m5C) modifications in human miRNAs. Our method uncovers that miRNAs undergo widespread cytosine modification in various sequence contexts.Furthermore, validation of our data with specific antibodies reveals both m5C and hm5C residues in human mature miRNAs. BS-miRNA-seq and MAmBA may contribute to the precise mapping of (h)m5C on miRNAs in various cell types and tissues, a key achievement toward2021-01-01T00:00:00Z2021 Dec2025-04-04T12:56:36.639Z2025-04-04T12:56:36.639ZS-EPMC86482623398013310.1080/15476286.2021.1927423