Abstract
sRNA-Seq is an unbiased method that allows for the discovery of small noncoding RNAs in bacterial transcriptomes through direct cloning and massively parallel sequencing by synthesis. Small bacterial transcripts are enriched from a total RNA preparation and modified with 5′ and 3′ linkers that allow for downstream amplification and sequencing. This protocol includes a treatment that depletes small RNA fractions of tRNAs and 5S rRNA, thereby enriching the starting pool for non-tRNA/rRNA sequences. This protocol can be readily modified to target different RNA species for depletion or to change the size range of RNAs to be sequenced. Thus, sRNA-Seq represents a comprehensive, versatile cloning protocol that may be applicable to the cloning of small RNAs of any size range from any organisms.
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Acknowledgments
We thank Kip Bodi for writing the script for designing tRNA-depletion oligonucleotides. This work was supported by Award Number K12GM074869 (J.M.L.) and A145746 (A.C.) from the National Institute of General Medical Sciences and National Institute of Health, respectively. A.C. is a Howard Hughes Medical Institute investigator. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute Of General Medical Sciences or the National Institutes of Health.
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Liu, J.M., Camilli, A. (2011). Discovery of Bacterial sRNAs by High-Throughput Sequencing. In: Kwon, Y., Ricke, S. (eds) High-Throughput Next Generation Sequencing. Methods in Molecular Biology, vol 733. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-089-8_5
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DOI: https://doi.org/10.1007/978-1-61779-089-8_5
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