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, dcl1-7 and PTH-AGO1 transgenic plants 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. One gram of wild type (WT-IP) or PTH-AGO1 (AGO1-IP) transgenic plant leaves was ground in liquid nitrogen and dissolved in 5 mL lysis buffer (50mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 5 mM DTT and protease inhibitor). After centrifugation, supernatants were incubated with IgG Sepharose 6 Fast Flow agarose beads (GE Healthcare) and rotated for 1 h at 4 °C. The beads were washed five times with lysis buffer. RNA in the immunoprecipitates was recovered with TRIzol reagent (Ambion). Total RNA or IP RNA 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 by Illumina HiSeq2000. For total RNA from wild type and dcl1-7 plants, small RNA libraries were made independently from two biological replicates. WT-IP, AGO1-IP, and its input libraries were made independently from three biological replicates.