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. 2020 Jan;17(1):23-32.
doi: 10.1080/15476286.2019.1664250. Epub 2019 Sep 16.

LOTTE-seq (Long Hairpin Oligonucleotide Based tRNA High-Throughput Sequencing): Specific Selection of tRNAs With 3'-CCA End for High-Throughput Sequencing

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LOTTE-seq (Long Hairpin Oligonucleotide Based tRNA High-Throughput Sequencing): Specific Selection of tRNAs With 3'-CCA End for High-Throughput Sequencing

Lieselotte Erber et al. RNA Biol. .
Free PMC article

Abstract

Transfer RNAs belong to the most abundant type of ribonucleic acid in the cell, and detailed investigations revealed correlations between alterations in the tRNA pool composition and certain diseases like breast cancer. However, currently available methods do not sample the entire tRNA pool or lack specificity for tRNAs. A specific disadvantage of such methods is that only full-length tRNAs are analysed, while tRNA fragments or incomplete cDNAs due to RT stops at modified nucleosides are lost. Another drawback in certain approaches is that the tRNA fraction has to be isolated and separated from high molecular weight RNA, resulting in considerable labour costs and loss of material. Based on a hairpin-shaped adapter oligonucleotide selective for tRNA transcripts, we developed a highly specific protocol for efficient and comprehensive high-throughput analysis of tRNAs that combines the benefits of existing methods and eliminates their disadvantages. Due to a 3'-TGG overhang, the adapter is specifically ligated to the tRNA 3'-CCA end. Reverse transcription prior to the ligation of a second adapter allows to include prematurely terminated cDNA products, increasing the number of tRNA reads. This strategy renders this approach a powerful and universal tool to analyse the tRNA pool of cells and organisms under different conditions in health and disease.

Keywords: CCA end; LOTTE-seq; high-throughput sequencing of tRNA; mature tRNA; tRNA adapter ligation; tRNA pool; tRNA-seq.

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Grant support

This work was supported by the Deutsche Forschungsgemeinschaft under grant numbers STA 850/27-1 and MO 634/13-1 to PFS and MM (under the auspices of SPP 1784) and by the Bundesministerium für Bildung und Forschung under grant number BMBF 031A538A (de.NBI/RBC) to PFS;Bundesministerium für Bildung und Forschung [BMBF 031A538A]; Deutsche Forschungsgemeinschaft [MO 634/13-1]; Deutsche Forschungsgemeinschaft [STA 850/27-1].

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