Experimental and computational workflow for the analysis of tRNA pools from eukaryotic cells by mim-tRNAseq

Andrew Behrens; Danny D Nedialkova

doi:10.1016/j.xpro.2022.101579

Experimental and computational workflow for the analysis of tRNA pools from eukaryotic cells by mim-tRNAseq

STAR Protoc. 2022 Jul 31;3(3):101579. doi: 10.1016/j.xpro.2022.101579. eCollection 2022 Sep 16.

Authors

Andrew Behrens¹, Danny D Nedialkova²

Affiliations

¹ Mechanisms of Protein Biogenesis, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
² Mechanisms of Protein Biogenesis, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; Department of Chemistry, Technical University of Munich, 85748 Garching, Germany. Electronic address: nedialkova@biochem.mpg.de.

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).

Keywords: Bioinformatics; Gene Expression; Molecular Biology; RNAseq; Sequencing; Systems biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular
Eukaryotic Cells* / metabolism
Protein Biosynthesis
RNA, Transfer* / genetics
Workflow

Substances

RNA, Transfer