Identification of Phloem Cell Transcriptome via Single-Cell RNA-Sequencing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for dissecting cellular heterogeneity in plants at a high resolution. In particular, scRNA-seq has been applied to unravel the complex cell identities and regulatory networks of distinct phloem cell types. Because phloem cells are small and under high osmotic pressure, isolating and maintaining viable protoplasts is a challenge for successful phloem scRNA-seq. Here, we present an optimized protocol for isolating vasculature-enriched protoplasts from mature Arabidopsis thaliana leaves. Following tissue harvesting, protoplasting is performed using a cocktail of cell wall-degrading enzymes suspended in an osmotically stabilized buffer. The resulting cell suspension is purified by removing debris through subsequent wash steps and validated using phloem-specific markers. Besides providing a reliable and reproducible method for isolating high-purity phloem-enriched protoplasts, we introduce the basic pipeline for downstream analysis.