Transcriptome analysis using RNA-Seq.

Wieteke A M Hoeijmakers, Richárd Bártfai, Hendrik G Stunnenberg

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

Methods in molecular biology (Clifton, N.J.) 2013

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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.

Citation

Wieteke A M Hoeijmakers, Richárd Bártfai, Hendrik G Stunnenberg. Transcriptome analysis using RNA-Seq. Methods in molecular biology (Clifton, N.J.). 2013;923:221-39