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| Status |
Public on Jul 01, 2019 |
| Title |
High-throughput sequencing of Pol IV and RDR2 transcripts, and their DCL3-diced products, generated from an M13mp18 (+) strand DNA template |
| Organism |
synthetic construct |
| Experiment type |
Other
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| Summary |
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.
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| 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.
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| Contributor(s) |
Wang F, Singh J, Pikaard CS |
| Citation(s) |
31398324 |
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| Submission date |
Feb 05, 2019 |
| Last update date |
Sep 30, 2019 |
| Contact name |
Craig S. Pikaard |
| E-mail(s) |
cpikaard@indiana.edu
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| Organization name |
Howard Hughes Medical Institute, Indiana University
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| Department |
Department of Biology and Department of Molecular and Cellular Biochemistry
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| Street address |
915 E. Third Street
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| City |
Bloomington |
| State/province |
Indiana |
| ZIP/Postal code |
47405 |
| Country |
USA |
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| Platforms (1) |
| GPL19424 |
Illumina NextSeq 500 (synthetic construct) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA521002 |
| SRA |
SRP183693 |
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