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| Status |
Public on Jul 01, 2020 |
| Title |
A universal, benchtop, gel-free method for the rapid and simultaneous isolation of all known classes of functional small RNAs |
| Organisms |
Arabidopsis thaliana; Drosophila melanogaster; Mus musculus |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
All known silencing small (s)RNAs operate via ARGONAUTE(AGO)-family proteins within RNA-induced-silencing-complexes (RISCs). Based on AGOs conserved biochemical properties, we have developed a universal, 15-min benchtop extraction procedure allowing simultaneous purification of all classes of RISC-associated sRNAs known to date, without prior knowledge of the samples-intrinsic AGO repertoires. Optimized into a user-friendly kit, the method –coined “TraPR” for Trans-kingdom, rapid, affordable Purification of RISCs– operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, including from minute amounts of input material. The method is highly suited for direct sRNA deep-sequencing, with TrAPR-generated libraries being qualitatively and quantitatively at least on-par with those obtained via gold-standard procedures involving tedious polyacrylamide gel excisions. TraPR considerably improves the quality and consistency of sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots and mammalian plasma, and regardless of RNA contaminants or samples’ RNA-degradation status.
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| Overall design |
Trans-kingdom, rapid, affordable Purification of RISC (TraPR) protocol for sRNA isolation was compared to standard sRNA library preparation from total RNA. A variety of source tissue, like Arabidopsis flower buds, Drosophila ovaries and mouse livers and plasa were used for these comaparisons.
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| Contributor(s) |
Oberlin S, Grentzinger T, Handler D, Brennecke J, Voinnet O |
| Citation(s) |
32496553 |
| Submission date |
Jul 18, 2019 |
| Last update date |
Jul 01, 2020 |
| Contact name |
Stefan Oberlin |
| E-mail(s) |
stefan.oberlin@ucsf.edu
|
| Organization name |
ETH Zürich
|
| Department |
Department of Biology
|
| Street address |
Universitätstrasse 2
|
| City |
Zürich |
| ZIP/Postal code |
8092 |
| Country |
Switzerland |
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| Platforms (4)
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| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
| GPL17275 |
Illumina HiSeq 2500 (Drosophila melanogaster) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
| GPL19580 |
Illumina NextSeq 500 (Arabidopsis thaliana) |
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| Samples (49)
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| Relations |
| BioProject |
PRJNA555390 |
| SRA |
SRP215328 |
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