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2'-O-methylation (Nm) is pivotal in rRNA and the internal site of mRNA. Here, we present an integrated bioinformatics protocol for the identification of 2'-O-methylation using nanopore direct RNA sequencing (RNA-seq) data. We describe steps for software installation, data collection, and training the machine learning model. We then detail procedures for mapping 2'-O-methylation in both rRNA and mRNA in yeast and human cells. For complete details on the use and execution of this protocol, please refer to Li et al..