Target-Enrichment Sequencing for Detailed Characterization of Small RNAs. Mus musculus

Identification of important, functional small RNA (sRNA) species is presently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method termed Target-Enrichment of small RNAs (TEsR), which 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 RNA, removal of non-targeted 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, and allows the detection of the complete sequence (including sequence variants, 5’ and 3’ ends) of precursors, intermediate and mature forms, and all this quantitatively. Overall design: RNA extracted from mouse embryonic fibroblast cell lines (MEFs), induced or uninduced for TRF2 conditional knock out, were used for targeted enrichment sequencing of small RNA (TEsR) and MiSeq sequencing or standard TruSeq small RNA sequencing. Each treatment had at least two replicates.