small RNA sequencing from leaf of Arabidopsis to identify tsRNA

tsRNA is newly found small non-coding RNA with important biological function. However, the knowlede of diversity, biogeneis and function of tsRNA in plant is still lacking. Here, we selected 10-60 nt small RNA for high-throughput sequencing and uncovered the diversity,biogenesis and potentical function of tsRNA in Arabidopsis. Col-0, rns1 and rns2 were grown under long-day (16h light) condition at 23°C. Leaves of four week old plants were harvested 3 h after wounding, or as control leaves without wounding. As for in vitro tRNAs cleavage assay, 0.5 μg tRNAs were incubated at 20°C in the presence of 15 μL leaf enzymatic extract for 30 min in a 90 μL reaction volume complemented with DEPC-H2O. As negative controls, 0.5 μg tRNAs in the absence of leaf enzymatic extract or 15 μL leaf enzymatic extract without additional tRNAs were incubated under the same conditions, respectively. After incubation, RNAs were extracted by TRIzol regent and separated on a 15% TBE-urea polyacrylamide gel.Total RNA or RNA underwent in vitro cleavage was fractionated by electrophoresis. RNA from 10 to 60 nt was sliced and recovered. The purified small RNA was subjected to adapter ligation, reverse transcription, PCR amplification with NEXTflex Small RNA Sequencing Kit (Bioo Scientific), and the resulting libraries sent for deep sequencing.