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Genome-wide identification of transcript start and end sites by transcript isoform sequencing

Nature Protocols volume 9, pages 1740–1759 (2014)Cite this article

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Abstract

Hundreds of transcript isoforms with varying boundaries and alternative regulatory signals are transcribed from the genome, even in a genetically homogeneous population of cells. To study this transcriptional heterogeneity, we developed transcript isoform sequencing (TIF-seq), a method that allows the genome-wide profiling of full-length transcript isoforms defined by their exact 5′ and 3′ boundaries. TIF-seq entails the generation of full-length cDNA libraries, followed by their circularization and the sequencing of the junction fragments spanning the 5′ and 3′ transcript ends. By determining the respective co-occurrence of start and end sites of individual transcript molecules, TIF-seq can distinguish variations that conventional approaches for mapping single ends cannot, such as short abortive transcripts, bicistronic messages and overlapping transcripts that differ in lengths. The TIF-seq protocol we describe here can be applied to any eukaryotic organism (e.g., yeast, human), and it requires 6–10 d for generating TIF-seq libraries, 10 d for sequencing and 2–3 d for analysis.

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Figure 1: Detailed experimental workflow of TIF-seq.
Figure 2: TIF-seq quality controls and anticipated results.
Figure 3: Gel size-selection of long full-length cDNA.

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Acknowledgements

We thank R. Aiyar for help in editing and refining the manuscript; A.I. Järvelin, J. Zaugg and S. Clauder-Münster for help in optimizing the TIF-seq protocol; and the members of the Steinmetz laboratory for helpful discussions and critical comments. This study was technically supported by the EMBL Genomics Core Facility. This study was financially supported by the US National Institutes of Health and the EMBL (to L.M.S.).

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Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany

    Vicent Pelechano, Wu Wei, Petra Jakob & Lars M Steinmetz

  2. Stanford Genome Technology Center, Palo Alto, California, USA

    Wu Wei & Lars M Steinmetz

  3. Department of Genetics, Stanford University School of Medicine, Stanford, California, USA

    Lars M Steinmetz

Authors
  1. Vicent Pelechano
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  2. Wu Wei
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  3. Petra Jakob
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  4. Lars M Steinmetz
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Contributions

W.W., V.P. and L.M.S. conceived the project; V.P. developed the TIF-seq method; P.J. contributed to further method optimization; W.W. and V.P. performed data analysis; and L.M.S. supervised the study. All authors wrote the manuscript.

Corresponding author

Correspondence to Lars M Steinmetz.

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The authors declare no competing financial interests.

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Pelechano, V., Wei, W., Jakob, P. et al. Genome-wide identification of transcript start and end sites by transcript isoform sequencing. Nat Protoc 9, 1740–1759 (2014). https://doi.org/10.1038/nprot.2014.121

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