An optimized FACS-free single-nucleus RNA sequencing (snRNA-seq) method for plant science research. (January 2023)

• Record Type: Journal Article
• Authors: Wang, Kaimeng; Zhao, Caiyao; Xiang, Sunhuan; Duan, Kunyu; Chen, Xiaoli; Guo, Xing; Sahu, Sunil Kumar
• Is Part Of: Plant science. Volume 326(2023)
• Publication Date: 2023-01
• Subjects: Arabidopsis thaliana -- Mature leaves -- ScRNA-seq -- SnRNA-seq; Botany -- Periodicals; Botanique -- Périodiques; 580
• DOI: 10.1016/j.plantsci.2022.111535
• Languages: English
• ISSNs: 0168-9452

Abstract

Abstract: Recently, single-cell RNA sequencing (scRNA-seq) provides unprecedented power for accurately understanding gene expression regulatory mechanisms. However, scRNA-seq studies have limitations in plants, due to difficulty in protoplast isolation that requires enzymatic digestion of the cell walls from various plant tissues. Therefore, to overcome this problem, we developed a nuclei isolation approach that does not rely on Fluorescence Activated Cell Sorting (FACS). We validated the robustness of the FACS-free single-nucleus RNA sequencing (snRNA-seq) methodology in mature Arabidopsis plant tissue by comparing it to scRNA-seq results based on protoplasts extracted from the same batch of leaf materials. Sequencing results demonstrated the high quality of snRNA-seq data, as well as its utility in cell type classification and marker gene identification. This approach also showed several advantages, including the ability to use frozen samples, taking less suspension preparation time, and reducing biased cellular coverage and dissociation-induced transcriptional artifacts. Surprisingly, snRNA-seq detected two epidermal pavement cell clusters, while scRNA-seq only had one. Furthermore, we hypothesized that these two epidermal cells represent the top and lower epidermis based on differences in expression patterns of cluster-specific expressed genes. In summary, this study has advanced the application of snRNA-seq in Arabidopsis leaves and confirmed the advantages of snRNA-seq in plant research. Graphical Abstract: ga1 Highlights: Developed Fluorescence Activated Cell Sorting (FACS)-free nuclei isolation method. Validated the robustness of snRNA-seq methodology in mature Arabidopsis leaves. Performed comparative analysis between snRNA-seq versus scRNA-seq data. snRNA-seq enabled cell type classification and marker gene identification. Method is applicable to frozen samples with short preparation time.