Tn5 tagments and transposes oligos to single-stranded DNA for strand-specific RNA sequencing
- 1Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China;
- 2Department of Medical Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China
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↵3 These authors contributed equally to this work.
Abstract
Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid substrates for the Tn5 transposon must be explored to increase the applications of Tn5. Here, we found that the Tn5 transposon can transpose oligos into the 5′ end of single-stranded DNA longer than 140 nucleotides. Based on this property of Tn5, we developed a tagmentation-based and ligation-enabled single-stranded DNA sequencing method called TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we applied TABLE-seq to strand-specific RNA sequencing, starting with as little as 30 pg of total RNA. Moreover, compared with traditional dUTP-based strand-specific RNA sequencing, this method detects more genes, has a higher strand specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.277213.122.
- Received August 18, 2022.
- Accepted February 1, 2023.
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