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. 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