Tissue-Specific High-Quality Protoplast Isolation for Single-Cell Analyses in Arabidopsis

Single-cell RNA sequencing (scRNAseq) has become an essential tool in plant biology to gain high-resolution information on cell type-specific gene expression, developmental dynamics, and tissue-specific responses to environmental factors. A major technical challenge in applying scRNAseq to plants is the efficient and reproducible isolation of viable single cells without cell walls, representing the heterogeneity of plant tissues. Protoplast isolation, which removes the cell wall enzymatically to yield single, living plant cells, is a widely used approach for this purpose. However, successful protoplast isolation requires careful optimization to maintain cell viability and preserve cell-type-specific transcriptome profiles. Here, we present a robust and reproducible protocol for isolating high-quality protoplasts from multiple Arabidopsis thaliana tissues, including above-ground (leaves, stems, flowers) and root tissues. This protocol accounts for the unique physiological properties of tissue types, optimizing enzymatic digestion conditions and purification steps to ensure high yield and broad representation of diverse cell types. The resulting protoplasts are suitable for applications such as scRNAseq, flow cytometry (FACS), and transient gene expression analysis. It allows for the generation of single-cell suspensions from multiple tissue types. The presented method supports exploration of cellular heterogeneity and enhances the reliability of single-cell approaches in plant research.