Efficient and quantitative high-throughput tRNA sequencing

• Published in: Nature methods Vol. 12; no. 9; pp. 835 - 837
• Main Authors: Zheng, Guanqun, Qin, Yidan, Clark, Wesley C, Dai, Qing, Yi, Chengqi, He, Chuan, Lambowitz, Alan M, Pan, Tao
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-09-2015; Nature Publishing Group
• Subjects: 101/58; 45/23; 45/77; 45/90; 45/91; 631/1647/2017/2003; 631/1647/514/1949; 631/337/572; 82/80; 82/83; Algorithms; Base Sequence; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical Engineering/Biotechnology; brief-communication; Cellular biology; DNA methylation; Gene Library; Genetic research; HEK293 Cells; High-Throughput Nucleotide Sequencing - methods; Humans; Life Sciences; Methods; Molecular Sequence Data; Proteomics; Ribonucleic acid; RNA; RNA sequencing; RNA, Transfer - genetics; Transfer RNA
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.3478

The combination of an engineered demthylase and a highly processive reverse transcriptase during tRNA library preparation for high-throughput sequencing allows comprehensive profiling of the small RNAs.
Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.