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 m g 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.