Abstract
Scan RNAs (scnRNAs) are developmentally regulated siRNAs of ~26–32 nucleotides in length that are involved in programmed DNA elimination in Tetrahymena. scnRNAs are loaded onto the Piwi-related protein Twi1p and 2′-O-methylated at their 3′ termini. We describe two alternative strategies for analyzing the Twi1p-loaded scnRNAs: preparation of loaded scnRNAs by immuno-purification of the Twi1p–scnRNA complex and exclusion of non-methylated scnRNAs during cDNA library construction using periodate oxidation.
The original online version for this Chapter can be found at http://dx.doi.org/10.1007/978-1-62703-694-8_20
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-62703-694-8_20
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Acknowledgments
We thank Julius Brennecke and his laboratory for providing a small RNA sequencing protocol and for technical advice. Solexa sequencings were performed at the Next Generation Sequencing (NGS) unit of the Campus Science Support Facilities (CSF) (http://csf.ac.at). Our research was supported by an ERC Starting Grant (204986) under the European Community’s Seventh Framework Programme, by the Special Research Program (SFB) “RNA regulation of the transcriptome” (F4307-B09) from the Austrian Science Fund (FWF), and by core funding from the Austrian Academy of Sciences to KM.
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Noto, T., Kurth, H.M., Mochizuki, K. (2014). Analysis of Piwi-Loaded Small RNAs in Terahymena . In: Siomi, M. (eds) PIWI-Interacting RNAs. Methods in Molecular Biology, vol 1093. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-694-8_17
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DOI: https://doi.org/10.1007/978-1-62703-694-8_17
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