High-throughput RNA sequencing (RNA-Seq) has quickly occupied center stage in the repertoire of available tools for transcriptomics. Among many advantages, the single-nucleotide resolution of this powerful approach allows mapping on a genome-wide scale of splice junctions and polyadenylation sites, and thus, the precise definition of mature transcript boundaries. This greatly facilitated the transcriptome annotation of the human pathogen Trypanosoma brucei, a protozoan organism in which all mRNA molecules are matured by spliced leader (SL) trans-splicing from longer polycistronic precursors. The protocols described here for the generation of three types of libraries for Illumina RNA-Seq, 5′-SL enriched, 5′-triphosphate-end enriched, and 3′-poly(A) enriched, enabled the discovery of an unprecedented heterogeneity of pre-mRNA-processing sites, a large number of novel coding and noncoding transcripts from previously unannotated genes, and quantify the cellular abundance of RNA molecules. The method for producing 5′-triphosphate-end-enriched libraries was instrumental for obtaining evidence that transcription initiation by RNA polymerase II in trypanosomes is bidirectional and biosynthesis of mRNA precursors is primed not only at the beginning of unidirectional gene clusters, but also at specific internal sites.
Ingolia NT, Ghaemmaghami S, Newman JR et al (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218–223CrossRefPubMedCentralPubMedGoogle Scholar
Cui P, Lin Q, Ding F et al (2010) A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing. Genomics 96:259–265CrossRefPubMedGoogle Scholar
White TC, Rudenko G, Borst P (1986) Three small RNAs within the 10 kb trypanosome rRNA transcription unit are analogous to domain VII of other eukaryotic 28S rRNAs. Nucleic Acids Res 14:9471–9489CrossRefPubMedCentralPubMedGoogle Scholar
Siegel TN, Hekstra DR, Wang X et al (2010) Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites. Nucleic Acids Res 38:4946–4957CrossRefPubMedCentralPubMedGoogle Scholar