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. Overall design: 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.

tsRNA是新发现的具有重要生物学功能的小型非编码RNA（small non-coding RNA），但目前学界对植物中tsRNA的多样性、生物发生（biogenesis）与功能的认知仍较为匮乏。本研究选取10~60 nt的小型RNA开展高通量测序，揭示了拟南芥（Arabidopsis）中tsRNA的多样性、生物发生过程及潜在功能。
实验设计详情如下：将Col-0、dcl1-7及PTH-AGO1转基因植株置于长日照（16 h光照）条件下，于23℃培养。待植株生长至4周时，收集创伤处理3 h后的叶片，同时以未施加创伤处理的叶片作为对照。取1 g野生型（WT-IP）或PTH-AGO1（AGO1-IP）转基因植株的叶片，在液氮中研磨后加入5 mL裂解缓冲液（pH 7.5的50 mM Tris-HCl、150 mM NaCl、5 mM MgCl₂、0.5% NP-40、5 mM DTT及蛋白酶抑制剂）。离心后取上清，与IgG Sepharose 6 Fast Flow琼脂糖微球（GE Healthcare）混合，于4℃旋转孵育1 h。随后用裂解缓冲液洗涤微球5次，免疫沉淀产物中的RNA采用TRIzol试剂（Ambion）回收。将总RNA或免疫沉淀RNA通过电泳分级分离，切取10~60 nt区间的RNA片段并回收纯化。将纯化得到的小型RNA依次进行接头连接、反转录及PCR扩增，所用试剂盒为NEXTflex Small RNA Sequencing Kit（Bioo Scientific），构建完成的文库通过Illumina HiSeq2000进行深度测序。其中，野生型与dcl1-7植株的总RNA小型RNA文库，设置2次生物学重复独立构建；WT-IP、AGO1-IP及其输入样本的文库，均设置3次生物学重复独立构建。