Transcriptome analysis using RNA-Seq

Methods Mol Biol. 2013;923:221-39. doi: 10.1007/978-1-62703-026-7_15.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Profiling / methods*
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Plasmodium / genetics*
  • RNA, Protozoan / chemistry
  • RNA, Protozoan / genetics
  • RNA, Protozoan / isolation & purification
  • Sequence Analysis, RNA / methods*
  • Transcriptome*

Substances

  • RNA, Protozoan