Abstract
BackgroundIdentifying 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.ResultsBy 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.ConclusionThis protocol simplifies and facilitates generation of DNase-seq libraries from plant tissues for high resolution mapping of DNase I hypersensitive sites.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0087-1) contains supplementary material, which is available to authorized users.
Highlights
Identifying cis-regulatory elements is critical in understanding the direct and indirect regulatory mechanisms of gene expression
We found that we were unable to produce sufficient quantities of DNAse I digested DNA for NextGen sequencing using a similar version of this protocol when processing Arabidopsis root tissue samples
The enormous required volume of preparations was prohibitive for the embedding of sufficient amounts of nuclei into the constricted volume of a pulse field gel electrophoresis (PFGE) agarose plug
Summary
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. Cis-regulatory elements (CREs) are short DNA sequences which are used by regulatory proteins such as transcription factors (TFs) to control the expression of genes [1] Because these elements need to be physically accessible to their respective regulatory proteins, they are often found in regions of the genome known as ‘open chromatin’ that are either unbound by or depleted of nucleosomes [1].
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