Abstract
Quantifying tRNAs is crucial for understanding how they regulate mRNA translation but is hampered by their extensive sequence similarity and premature termination of reverse transcription at multiple modified nucleotides. Here, we describe the use of modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which overcomes these limitations with optimized library construction and a comprehensive toolkit for data analysis and visualization. We outline algorithm improvements that enhance the efficiency and accuracy of read alignment and provide details on data analysis outputs using example datasets. For complete details on the use and execution of this protocol, please refer to Behrens et al. (2021).