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 in | Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 6; pp. 2886 - 2893 |
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| Main Authors | , , , , , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
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National Academy of Sciences
11.02.2020
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| Abstract | 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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| AbstractList | 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. RNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are desirable. All existing RNA sequencing methods rely on the conversion of RNA into double-stranded DNA through reverse transcription followed by second-strand synthesis. The latter step requires additional enzymes and purification, and introduces sequence-dependent bias. Here, we show that Tn5 transposase, which randomly binds and cuts double-stranded DNA, can directly fragment and prime the RNA/DNA heteroduplexes generated by reverse transcription. The primed fragments are then subject to PCR amplification. This provides an approach for simple and accurate RNA characterization and quantification. 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. 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.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. |
| Author | Wang, Guanbo Xie, X. Sunney Sun, Yue Oh, Juntaek Wang, Dong Huang, Yanyi Di, Lin Yao, Jiacheng Lee, Raymond W. Wang, Jianbin Lao, Kaiqin Xu, Jun Li, Jie Fu, Yusi Wu, Yalei Zheng, Genhua Liu, Lu |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/31988135$$D View this record in MEDLINE/PubMed |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 ObjectType-Article-2 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23 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) |
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| Snippet | Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA... RNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are... |
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| SubjectTerms | 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 |
| Title | RNA sequencing by direct tagmentation of RNA/DNA hybrids |
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