Small RNA sequencing on affinity capture-purified tRNA-derived small RNAs (tsRNAs)

During stress conditions, particular tRNAs are cleaved by stress-induced endonucleases resulting in distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide array of mostly stress-related biological processes, however how exactly they exert their functions mechanistically is still largely unknown. Since parental tRNAs carry post-transcriptional modifications, stress-induced tsRNAs are likely modified, too. In order to use endogenously modified tsRNAs, we developed a biochemical pipeline for the production and purification of specific tsRNAs using a human cell culture system. Ectopic expression of Angiogenin combined with chromatography of small RNAs resulted in the production of endogenously modified 5' tsRNAs on a preparative scale. Small RNA sequencing on purified tsRNAs was used to determine the identity and purity of the 5' tsRNA preparations. Overall design: HEK293 cells expressing human Angiogenin from an integrated via the Flp-In™-T-REx™ system were exposed to doxycycline for 48 hours, followed by chromatographic RNA purification for 5' tsRNA-GlyGCC and 5' tsRNA-GluCUC