Study : RNA-seq from roots of 14 day old col-0 Arabidopsis thaliana carrying INTACT


RNA-seq from roots of 14 day old col-0 Arabidopsis thaliana carrying INTACT
Background: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus the accessibility of these loci for TF binding is highly regulated and determines where and when TF bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana, and define organ-specific regulatory sites and binding motifs of transcription factors at these sites. Results: We coupled the recently described INTACT (Isolation of Nuclei TAgged in specific Cell Types) and ATAC-seq (Assay for Transposase-Accessible Chromatin with highthroughput sequencing) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis, based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions, but are correlated with root-specific expression of distant genes. Importantly these gene-distant sites are enriched for binding motifs of TFs important for root development, as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel genedistant regulatory elements. Conclusion: By coupling the INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity, possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions. Overall design: Roots were harvested from 14-day old seedlings from transgenic Arabidopsis thaliana col-0 roots, containing a single-plasmid INATCT system under the constitutive 35S promoter. RNA was extracted using TRIZOL (Life Technologies) reagent and cleaned with RNA Clean Concentratorâ„¢-5 (Zymo). Libraries were prepared for NGS sequencing with NEBNext RNA library prep kit (module E7530-E7490).


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