Cumbie Jason S, Filichkin Sergei A, Megraw Molly
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 USA.
Center for Genome Research and Biocomputing, Oregon State University, 2082 Cordley Hall, Corvallis, OR 97331 USA.
Plant Methods. 2015 Sep 4;11:42. doi: 10.1186/s13007-015-0087-1. eCollection 2015.
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.
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.
This protocol simplifies and facilitates generation of DNase-seq libraries from plant tissues for high resolution mapping of DNase I hypersensitive sites.
识别顺式调控元件对于理解基因表达的直接和间接调控机制至关重要。目前的方法包括DNase-seq，该技术将对非特异性核酸内切酶DNase I的敏感性与高通量测序相结合，以在全基因组范围内识别调控DNA区域。虽然这种方法最初是为人类细胞系开发的，但后来的改进使得植物组织的处理成为可能。在处理DNA含量低的顽固组织时，挑战仍然存在。
通过在DNase-seq中去除需要使用凝胶琼脂糖块的步骤，我们能够将执行该方案所需的时间显著减少至少2天，同时也使处理困难的植物组织成为可能。我们将这个简化的方案称为DNase I SIM（简化的核内方法）。我们能够使用DNase I SIM成功地为拟南芥的叶片和根组织创建DNase-seq文库。
该方案简化并促进了从植物组织生成DNase-seq文库，用于DNase I超敏位点的高分辨率定位。
