RNA sequencing by direct tagmentation of RNA/DNA hybrids

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 117; no. 6; pp. 2886 - 2893
Main Authors Di, Lin, Fu, Yusi, Sun, Yue, Li, Jie, Liu, Lu, Yao, Jiacheng, Wang, Guanbo, Wu, Yalei, Lao, Kaiqin, Lee, Raymond W., Zheng, Genhua, Xu, Jun, Oh, Juntaek, Wang, Dong, Xie, X. Sunney, Huang, Yanyi, Wang, Jianbin
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 11.02.2020
Subjects
Adapters
Biological Sciences
Chimera - genetics
Complementary DNA
Deoxyribonucleic acid
DNA
DNA - genetics
DNA biosynthesis
DNA fingerprinting
DNA sequencing
DNA, Complementary - genetics
Gene expression
Gene Library
HEK293 Cells
HeLa Cells
Humans
Hybrids
Reverse transcription
Ribonucleic acid
RNA
RNA - genetics
Sequence Analysis, RNA - methods
Single-Cell Analysis
Synthesis
Transposase
Transposases - metabolism
Tn5 transposase
RNA-seq
single cell
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Summary:Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.
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1L.D. and Y.F. contributed equally to this work.
Author contributions: Y.F., K.L., X.S.X., Y.H., and J.W. designed research; L.D., Y.F., Y.S., J.L., L.L., G.W., J.X., J.O., and D.W. performed research; L.D., Y.F., L.L., J.Y., Y.W., R.W.L., G.Z., Y.H., and J.W. analyzed data; and L.D., X.S.X., Y.H., and J.W. wrote the paper.
2Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.
Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved December 31, 2019 (received for review November 11, 2019)
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.1919800117