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Improved DNase‑seq protocol facilitates high resolution mapping of DNase I hypersensitive sites in roots in Arabidopsis thaliana

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https://ir.library.oregonstate.edu/concern/articles/4x51hk87m

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  • Background: Identifying cis-regulatory elements is critical in understanding the direct and indirect regulatory mechanisms of gene expression. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high throughput sequencing to identify regions of regulatory DNA on a genome-wide scale. While this method was originally developed for human cell lines, later adaptations made the processing of plant tissues possible. Challenges still remain in processing recalcitrant tissues that have low DNA content. Results: By removing steps requiring the use of gel agarose plugs in DNase-seq, we were able to significantly reduce the time required to perform the protocol by at least 2 days, while also making possible the processing of difficult plant tissues. We refer to this simplified protocol as DNase I SIM (for simplified in-nucleus method). We were able to successfully create DNase-seq libraries for both leaf and root tissues in Arabidopsis using DNase I SIM. Conclusion: This protocol simplifies and facilitates generation of DNase-seq libraries from plant tissues for high resolution mapping of DNase I hypersensitive sites.
  • This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by BioMed Central. The published article can be found at: http://www.plantmethods.com/. Additional files can be located online at: http://www.plantmethods.com/content/11/1/42/additional
  • Keywords: Open chromatin, DNase-seq, Roots, DNase I hypersensitive sites, Nuclei, Arabidopsis
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  • Cumbie, J. S., Filichkin, S. A., & Megraw, M. (2015). Improved DNase-seq protocol facilitates high resolution mapping of DNase I hypersensitive sites in roots in Arabidopsis thaliana. Plant Methods, 11(1), 42. doi:10.1186/s13007-015-0087-1
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  • 11
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  • 1
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  • This work was supported by the National Institutes of Health [GM097188 to MM]; and by startup funds from Oregon State University.
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file details CumbieJasonBotanyPlantPathImprovedDNaseseqProtocol.pdf 2017-07-26 Público Baixar