Transcriptome Analysis Using RNA-Seq
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.
Key wordsPlasmodium Plasmodium falciparum Transcriptome RNA-sequencing (RNA-seq) Next-generation sequencing (NGS) Linear amplification for deep sequencing (LADS)
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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