DORQ-seq: high-throughput quantification of femtomol tRNA pools by combination of cDNA hybridization and Deep sequencing.

Autor:	Kristen M; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany., Lander M; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany., Kilz LM; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany., Gleue L; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany., Jörg M; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany., Bregeon D; IBPS, Biology of Aging and Adaptation, Sorbonne Université, Paris 75252, France., Hamdane D; Laboratoire de Chimie des Processus Biologiques, CNRS-UMR 8229, Collège De France, Université Pierre et Marie Curie, 11 place Marcelin Berthelot, 75231 Paris, Cedex 05, France., Marchand V; Université de Lorraine, IMoPA UMR7365 CNRS-UL, BioPole, 54000 Nancy, France., Motorin Y; Université de Lorraine, IMoPA UMR7365 CNRS-UL, BioPole, 54000 Nancy, France.; Université de Lorraine, Epitranscriptomics and RNA Sequencing (EpiRNA-Seq) Core Facility, UAR2008 IBSLor (CNRS-UL)/US40 (INSERM), 54000 Nancy, France., Friedland K; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany., Helm M; Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2024 Oct 14; Vol. 52 (18), pp. e89.
DOI:	10.1093/nar/gkae765
Abstrakt:	Due to its high modification content tRNAs are notoriously hard to quantify by reverse transcription and RNAseq. Bypassing numerous biases resulting from concatenation of enzymatic treatments, we here report a hybrid approach that harnesses the advantages of hybridization-based and deep sequencing-based approaches. The method renders obsolete any RNAseq related workarounds and correction factors that affect accuracy, sensitivity, and turnaround time. Rather than by reverse transcription, quantitative information on the isoacceptor composition of a tRNA pool is transferred to a cDNA mixture in a single step procedure, thereby omitting all enzymatic conversations except for the subsequent barcoding PCR. As a result, a detailed tRNA composition matrix can be obtained from femtomolar amounts of total tRNA. The method is fast, low in cost, and its bioinformatic data workup surprisingly simple. These properties make the approach amenable to high-throughput investigations including clinical samples, as we have demonstrated by application to a collection of variegated biological questions, each answered with novel findings. These include tRNA pool quantification of polysome-bound tRNA, of tRNA modification knockout strains under stress conditions, and of Alzheimer patients' brain tissues. (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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