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Transcriptome Analysis Using RNA-Seq

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Malaria

Part of the book series: Methods in Molecular Biology ((MIMB,volume 923))

Abstract

Transcriptome analysis by next-generation sequencing (RNA-seq) allows investigation of a transcriptome at unsurpassed resolution. One major benefit is that RNA-seq is independent of a priori knowledge on the sequence under investigation, thereby also allowing analysis of poorly characterized Plasmodium species. Here we provide a detailed protocol for RNA isolation and fragmentation, ribosomal RNA depletion, and cDNA synthesis that enables the preparation of a sequencing library from 1 to 2 μg of total RNA. Although we focus our discussion on the quantitative measurement of gene expression, this protocol is suited for many applications of RNA-seq and allows analysis of most RNA species.

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Acknowledgments

The research leading to these protocols has received funding from the Netherlands Organization for Scientific Research (ZonMw/NGI Horizon 93511023; NWO Toptalent 021.001.011) and the European Community (EVIMalaR EU-FP7_242095; ATLAS EU-FP7_221952). We would like to thank our colleagues Marjolein van Sluis, Maarten van der Velden, Arne Smits, Ulrike Jacobi, Hendrik Marks, Yan Tan, Eva Janssen-Megens, Adriana Salcedo-Amaya, and Kees-Jan Françoijs for valuable discussions and/or involvement in the development of this protocol. Finally, we would like to thank PlasmoDB (40) and GeneDB (41) for providing genomic resources for Plasmodium.

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Authors and Affiliations

  1. Department of Molecular Biology, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands

    Wieteke A. M. Hoeijmakers, Richárd Bártfai & Hendrik G. Stunnenberg

Authors
  1. Wieteke A. M. Hoeijmakers
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  2. Richárd Bártfai
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  3. Hendrik G. Stunnenberg
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Corresponding author

Correspondence to Hendrik G. Stunnenberg .

Editor information

Editors and Affiliations

  1. , Malaria Biology and Genetics Unit, Institut Pasteur, Rue du Dr Roux 28, Paris, 75724, France

    Robert Ménard

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Hoeijmakers, W.A.M., Bártfai, R., Stunnenberg, H.G. (2012). Transcriptome Analysis Using RNA-Seq. In: Ménard, R. (eds) Malaria. Methods in Molecular Biology, vol 923. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-026-7_15

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  • DOI: https://doi.org/10.1007/978-1-62703-026-7_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-025-0

  • Online ISBN: 978-1-62703-026-7

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