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Construction of Trypanosoma brucei Illumina RNA-Seq Libraries Enriched for Transcript Ends

  • Nikolay G. KolevEmail author
  • Elisabetta Ullu
  • Christian Tschudi
Part of the Methods in Molecular Biology book series (MIMB, volume 1201)

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.

Key words

Trypanosoma brucei RNA-Seq 5′-SL enriched 5′-Triphosphate-end enriched 3′-Poly(A) enriched Terminator exonuclease 

Notes

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Nikolay G. Kolev
    • 1
    Email author
  • Elisabetta Ullu
    • 2
    • 3
  • Christian Tschudi
    • 4
  1. 1.Department of Epidemiology of Microbial Diseases, School of Public HealthYale UniversityNew HavenUSA
  2. 2.Department of Internal Medicine, School of MedicineYale UniversityNew HavenUSA
  3. 3.Department of Cell Biology, School of MedicineYale UniversityNew HavenUSA
  4. 4.Department of Epidemiology of Microbial DiseasesYale School of Public HealthNew HavenUSA

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