Abstract
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.
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Acknowledgments
Work in our laboratory was funded in part by National Institute of Allergy and Infectious Diseases Grants AI28798 to EU and AI43594 and AI078333 to CT.
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Kolev, N.G., Ullu, E., Tschudi, C. (2015). Construction of Trypanosoma brucei Illumina RNA-Seq Libraries Enriched for Transcript Ends. In: Peacock, C. (eds) Parasite Genomics Protocols. Methods in Molecular Biology, vol 1201. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1438-8_9
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DOI: https://doi.org/10.1007/978-1-4939-1438-8_9
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-1438-8
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