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, 12 (9), 835-837

Efficient and Quantitative High-Throughput tRNA Sequencing

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Efficient and Quantitative High-Throughput tRNA Sequencing

Guanqun Zheng et al. Nat Methods.

Abstract

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.

Figures

Figure 1
Figure 1. Demethylase-thermostable group II intron RT tRNA sequencing (DM-TGIRT-seq)
(a) Our method differs from standard RNA-seq in two ways. First, a demethylase mixture was used to remove m1A, m1G and m3C modifications located at the Watson-Crick face. Second, a thermostable group II intron RT (TGIRT) was used that is less sensitive to tRNA structure and adds RNA-seq adaptors by template-switching without RNA ligation. (b) Demethylation efficiency for total tRNA as measured by QQQ LC-MS. (c) RT reaction for both purified tRNA and total RNA as template with (+) or without (−) demethylase treatment. Blue line shows the gel region excised for library construction.
Figure 2
Figure 2. Sequencing results
(a) 5’ position sequencing plot of a LeuAAG tRNA containing an m1G37 modification. (b) 5’ position sequencing plot of a GlnTTG tRNA containing an m1G9 modification. For panels (a) and (b), m1A58 stops correspond to very short DNA fragments that are outside the range of our sequenced cDNA, and hence cannot be visualized here. (c) As expected, tRNA isoacceptor expression in HEK293T cells does not correlate with the gene copy number, since human tRNA expression is tissue specific. (d) Comparison of the array fluorescent signals and sequencing reads for the Arg-tRNA family; error bar, n = 4 ± SD. (e) Mismatch and stops with and without demethylase treatment. Shown are modification positions of m1A58 in ValCAC, m3C32 in ThrAGT, m1G37 in ProTGG, and m1G9 in GlnCTG. Star indicates stops for ValCAC corresponding to very short reads that were not determined in this experiment. n = 4 ± SD. (f) Sequence logo at the same modified positions as in (e), centered at the modified residue. Untreated (−) is at the top and the demethylases treated (+) is at the bottom.

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