Efficient and quantitative high-throughput tRNA sequencing

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.