Preparation of enzymes and libraries for MapID-tRNA-seq to identify chemical modifications in human tRNAs

Abstract

Chemical modifications are abundant in tRNAs and play essential roles in tRNA biology and human diseases. We recently reported MapID-tRNA-seq that allows identification of chemical modifications in human tRNAs such as m¹A and m³C. MapID-tRNA-seq utilizes an evolved reverse transcriptase (RT-1306) that reads through and generates mutation signatures at m¹A and m³C modifications, which allows robust detection and semi-quantification of m¹A and m³C in human tRNAs. In addition, we developed MapIDs to consolidate the sequence redundancy within the human tRNA genome, with explicit annotations of genetic variance among highly similar tRNA genes. MapIDs help resolve a critical issue of false-positive discoveries of modifications caused by reads misalignment at genetic variance sites. In this chapter, we report detailed protocols for the in-house preparation and characterization of the two enzymes used in MapID-tRNA-seq library preparation (RT-1306 and the demethylase AlkB), tRNA-seq library preparation, and MapID-assisted sequencing analysis, to facilitate application and future development of the MapID-tRNA-seq method.

Keywords: AlkB; Chemical modifications; Human tRNAs; RT-1306; tRNA MapID; tRNA-seq.