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Method Article
Jay Shendure
University of Washington
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1516-1865
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. Here, we further develop and apply a novel method to assemble and functionally test libraries of greater than 2000 of the same putative enhancers as 192-mers, 354-mers, and 678-mers. We achieved a yield of 95% for 354-mers and 84% for 678-mers. Importantly, we observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.
Keywords
MPRA, enhancers, promoters, gene expression, gene regulation, reporter assay
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- supptable.xlsx
Group specific primers
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An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Jay Shendure
University of Washington
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1516-1865
Version History
CURRENT STATUS: Posted
Version 1
Posted 12 Oct, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. Here, we further develop and apply a novel method to assemble and functionally test libraries of greater than 2000 of the same putative enhancers as 192-mers, 354-mers, and 678-mers. We achieved a yield of 95% for 354-mers and 84% for 678-mers. Importantly, we observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- supptable.xlsx
Group specific primers
Loading...