High-throughput sequencing of Pol IV and RDR2 transcripts, and their DCL3-diced products, generated from an M13mp18 (+) strand DNA template. High-throughput sequencing of Pol IV and RDR2 transcripts, and their DCL3-diced products, generated from an M13mp18 (+) strand DNA template

In plants, the biogenesis of 24 nt and 23 nt small interfering RNAs (siRNAs) requires NUCLEAR RNA POLYMERASE IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). We show that single-stranded M13 bacteriophage DNA can be used as a template for siRNA synthesis in vitro. Deep sequencing of RNAs produced from the in vitro reactions of Pol IV, RDR2 and DCL3 shows that Pol IV transcribes the DNA into first-strand RNAs which RDR2 then uses as templates to synthesize complementary second strands. These siRNA precursor transcripts made by Pol IV and RDR2 are mostly 30-50 nt. An untemplated 3' terminal nucleotide is a characteristic of RDR2 transcripts. DCL3 dicing of double-stranded precursor RNAs synthesized by Pol IV and RDR2 generates siRNAs that are mostly 24 nt, with a smaller population of 23 nt also produced. Overall design: Deep sequencing of siRNA precursors produced by in vitro activities of Pol IV-RDR2, and mature siRNAs produced by DCL3 with a single-stranded M13 bacteriophage DNA template. Briefly, we purified recombinant NUCLEAR RNA POLYMERASE IV (Pol IV) and recombinant RNA-dependent RNA POLYMERASE 2 from Arabidopsis thaliana tissues, and incubated them with single-stranded bacteriophage M13 DNA that serves as a template to synthesize small RNA precursors. These precursors were then processed by recombinant DCL3 proteins that were expressed in Sf9 insect cells to produce small RNAs in vitro. These precursors and small RNAs were subject to high-throughput sequencing. RNAs synthesized by purified recombinant Pol IV from an rdr2 mutant background were served as a control for the deep-sequencing experiment. Please note that the processed data is clean reads which are mappable to the M13 ssDNA template that we used as a template for in vitro transcription. To investigate the size distributions, sequence preferences/characteristics of small RNA as well as their precursors in this study, the clean reads that are derived from Pol IV RDR2 and DCL3 activities by using a ssDNA template would be important for readers to understand our conclusion. Therefore, read sequences, read sizes and strandedness are provided in the processed datasets.

在植物中，24 nt与23 nt小干扰RNA（small interfering RNAs, siRNAs）的生物发生依赖于核RNA聚合酶IV（NUCLEAR RNA POLYMERASE IV, Pol IV）、RNA依赖的RNA聚合酶2（RNA-DEPENDENT RNA POLYMERASE 2, RDR2）以及类Dicer 3（DICER-LIKE 3, DCL3）。本研究证实，单链M13噬菌体DNA可作为体外合成siRNA的模板。对Pol IV、RDR2与DCL3体外反应体系中生成的RNA进行深度测序（deep sequencing）后发现，Pol IV可将该DNA转录为第一链RNA，随后RDR2以该第一链RNA为模板合成互补的第二链RNA。由Pol IV与RDR2生成的siRNA前体转录本长度多介于30~50 nt之间。RDR2转录产物的典型特征是带有非模板依赖的3'末端核苷酸。Pol IV与RDR2合成的双链前体RNA经DCL3切割后，生成的siRNA多为24 nt，同时也会产生少量23 nt的siRNA。

实验整体设计：对以单链M13噬菌体DNA为模板，经Pol IV-RDR2体外活性生成的siRNA前体，以及经DCL3切割生成的成熟siRNA进行深度测序。

简要而言，本研究从拟南芥（Arabidopsis thaliana）组织中纯化得到重组核RNA聚合酶IV（Pol IV）与重组RNA依赖的RNA聚合酶2，并将其与作为合成小RNA前体模板的单链M13噬菌体DNA共同孵育。随后，利用在Sf9昆虫细胞中表达的重组DCL3蛋白对这些前体进行切割，以在体外生成小RNA。

随后对这些前体与小RNA进行高通量测序（high-throughput sequencing）。本研究还以从rdr2突变体背景中纯化得到的重组Pol IV合成的RNA作为深度测序实验的对照。

请注意，加工后数据为可比对至本研究体外转录所用M13单链DNA（single-stranded DNA, ssDNA）模板的洁净读段（clean reads）。

为探究本研究中小RNA及其前体的长度分布、序列偏好性与特征，读者可通过本研究中来自Pol IV、RDR2与DCL3以单链DNA为模板的活性所生成的洁净读段，更好地理解本研究结论。因此，加工后数据集提供了读段序列、读段长度与链特异性（strandedness）信息。