Abstract
High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap–based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes ∼10 d to implement.
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Acknowledgements
We thank M. Philipps, S. Vicaire and B. Jost from the IGBMC Microarray and Sequencing platform (France Génomique consortium—ANR-10-INBS-0009). M.M.P. and V.P. were supported as postdoctoral fellows of the Ligue Nationale Contre le Cancer. This work was supported by funds from the Alliance Nationale pour les Sciences de la Vie et de la Santé–Institut Thématique Multi-organismes Cancer–Institut National du Cancer (INCa) grant 'Epigenomics of breast cancer', EpiPCa, the Ligue National Contre le Cancer (to H.G.; Equipe Labellisée), the INCa and the European Community contract LSHC-CT-2005-518417 'EPITRON'. Support from the Agence Nationale de la Recherche (ANRT-07-PCVI-0031-01, ANR-10-LABX-0030-INRT and ANR-10-IDEX-0002-02) is also acknowledged.
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M.M.P. conceived and designed the RNA-trap-seq strategy and performed bioinformatics analysis. M.M.P., V.P. and C.E. performed experiments and optimized the final experimental pipeline. M.M.P., V.P. and H.G. wrote the manuscript.
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A European patent application, ‘Methods for sequencing and identifying RNAs’ (EP 14 305 822.0) has been filed by M.M.P. and H.G.
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Portal, M., Pavet, V., Erb, C. et al. TARDIS, a targeted RNA directional sequencing method for rare RNA discovery. Nat Protoc 10, 1915–1938 (2015). https://doi.org/10.1038/nprot.2015.120
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DOI: https://doi.org/10.1038/nprot.2015.120
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