Identification of important, functional small RNA (sRNA) species is currently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method, termed target-enrichment of sRNAs (TEsR), that enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full-length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNAs, removal of nontargeted sRNAs and sequencing. By this approach, target sRNAs can be enriched by a factor of 500–30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5′ cap or of secondary structures or abundance levels. Moreover, TEsR allows the detection of the complete sequence (including sequence variants, and 5′ and 3′ ends) of precursors, as well as intermediate and mature forms, in a quantitative manner. A well-trained molecular biologist can complete the TEsR procedure, from RNA extraction to sequencing library preparation, within 4–6 d.
We thank all P.C. and F.d.d.F. laboratory members, especially G. Pascarella, K. Hashimoto and A. Bonetti, for insightful discussions. F.d.d.F.'s laboratory is supported by Associazione Italiana per la Ricerca sul Cancro (application 12971), AriSLA (project 'DDRNA and ALS'), Associazione Italiana per la Ricerca sul Cancro (AIRC; application 12971), Worldwide Cancer Research (Association for International Cancer Research (AICR), Research Infrastructure Fund (RIF) 14-1331), Research EPIGEN, Fondazione Cariplo (grants 2014-1215 and 2014-0812), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, Fondazione Telethon (GGP12059), the Human Frontier Science Program (contract RGP 0014/2012) and a European Research Council advanced grant (322726). P.C.'s lab is supported by a Research Grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to the RIKEN Center for Life Science Technologies. J.A. is supported by Marie Curie Initial Training Networks (FP7 PEOPLE 2012 ITN (CodeAge project no: 316354)). F.R. is supported by Fondazione Italiana per la Ricerca sul Cancro (FIRC, application number 12476).
Integrated supplementary information
Supplementary Figures 1–8 and Supplementary Tables 1 and 2.