Abstract
The introduction of RNA sequencing (RNA-seq), as a direct result of rapid progression of next-generation sequencing technologies, has revolutionized the world of transcriptomics. It enables quantification of the complete set of RNA with all its isoforms in a given cell, in far greater accuracy than before. Here, we provide a step-by-step guide to the sample preparation in RNA-seq and an overview of the bioinformatic analysis that is required following the cDNA sequencing.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M et al (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344–1349
Wilhelm BT, Marguerat S, Watt S, Schubert F, Wood V, Goodhead I et al (2008) Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453:1239–1243
Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH et al (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133: 523–536
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628. doi:10.1038/nmeth.1226
Cloonan N, Forrest AR, Kolle G, Gardiner BB, Faulkner GJ, Brown MK et al (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5:613–619
ENCODE Project Consortium, Bernstein BD, Birney E, Dunham I, Green ED, Gunter C et al (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111
Wang K, Singh D, Zeng Z, Coleman SJ, Huang Y, Savich GL et al (2010) MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res 38:e178
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ et al (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106
Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31: 46–53
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652
Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X et al (2010) Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat Biotechnol 28:503–510
Tani H, Mizutani R, Salam KA, Tano K, Ijiri K, Wakamatsu A et al (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res 22:947–956
Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, Nammo T et al (2012) KIF5B-RET fusions in lung adenocarcinoma. Nat Med 18:375–377
Kim D, Salzberg SL (2011) TopHat-fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol 12:R72
Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218–223
Morin RD, O’Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu AL et al (2008) Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res 18:610–621
Zhao J, Ohsumi TK, Kung JT, Ogawa Y, Grau DJ, Sarma K et al (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40:939–953
Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141
Maekawa S (2014) Medical genome sciences, graduate school of frontier sciences, The University of Tokyo
Suzuki A (2014) Medical genome sciences, graduate school of frontier sciences, The University of Tokyo
Acknowledgments
We would like to thank the sequencing team at the Sugano laboratory for helpful advice in writing this manuscript. I would also like to thank Nobuyoshi Akimitsu on BRIC-seq. This work was supported by MEXT KAKENHI Grant Number 221S0002.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Maekawa, S., Suzuki, A., Sugano, S., Suzuki, Y. (2014). RNA Sequencing: From Sample Preparation to Analysis. In: Miyamoto-Sato, E., Ohashi, H., Sasaki, H., Nishikawa, Ji., Yanagawa, H. (eds) Transcription Factor Regulatory Networks. Methods in Molecular Biology, vol 1164. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0805-9_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0805-9_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0804-2
Online ISBN: 978-1-4939-0805-9
eBook Packages: Springer Protocols