Background

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) is a popular technology to map DNA accessible sites and to identify putative regulatory elements (Lu et al., 2017; Huang et al., 2021). The accessibility of these elements plays an important role in controlling gene expression because they are transcription factor-binding sites. One major limitation associated with the use of ATAC-seq technology is the cellular complexity of the sample used. When working at the entire organ level, the profile of chromatin accessibility cannot reveal the differential and unique contributions of each cell and cell type composing the organ. To overcome this challenge, recent studies successfully tested ATAC-seq technology on thousands of individual plant nuclei isolated from Arabidopsis thaliana roots and various Zea mays organs (Dorrity et al., 2021; Farmer et al., 2021; Marand et al., 2021).

The integration of this molecular information with RNA-seq datasets generated from isolated protoplasts and nuclei revealed the impact of chromatin accessibility and regulatory elements in controlling gene expression (Denyer et al., 2019; Jean-Baptiste et al., 2019; Nelms and Walbot, 2019; Ryu et al., 2019; Shulse et al., 2019; Zhang et al., 2019; Dorrity et al., 2021; Farmer et al., 2021; Liu et al., 2020; Marand et al., 2021). Therefore, single-cell/nucleus RNA-seq and single nuclei ATAC-seq (sNucATAC-seq or snATAC-seq) have recently changed our assessment of the molecular mechanisms controlling gene expression. These technologies will also contribute to a better understanding of gene function to control plant cell differentiation, development, and plant cell response to environmental stresses (Rich-Griffin et al., 2020; Shaw et al., 2021).

The SNucATAC-seq technology relies on the permeabilization of the membrane of isolated plant nuclei to enable the penetration of the Tn5 transposase, the tagmentation of the accessible genomic DNA sequences, and the construction of Illumina-compatible libraries. In this manuscript, we describe the methodology used to conduct sNucATAC-seq. This method complements the protocol describing the implementation of single nuclei RNA-seq technology on plant samples (Thibivilliers et al., 2020). Here, we describe a protocol to isolate and purify nuclei from model plants and crop species before permeabilizing the nuclear membrane to enable the penetration of the Tn5 transposase (Figure 1). Examples of successful sNucATAC-seq profiles from Arabidopsis thaliana and Glycine max are also provided.