Gene Expression Analysis from Specific Root Cell Types

In plant roots, the diversity of tissues along the radial axis and of developmental stages along the longitudinal axis implies a highly dynamic and heterogeneous gene expression. Many master regulators of gene expression exhibit distinctive temporal expression patterns or are restricted to specific cell types, making whole-root molecular analyses insufficient to resolve their roles in developmental and physiological processes. Here, we report a protocol for fluorescence-activated cell sorting (FACS)-coupled RNA sequencing to analyze, reconstruct, and ultimately dissect the gene regulatory networks (GRNs) involved in root development and physiology, particularly well-suited for genes that are expressed transiently or in rare or restricted cell types. The method relies on selecting an appropriate cell-type-specific reporter line, which enables the isolation of labeled cells with high specificity and minimal contamination. Furthermore, we discuss the relative advantages of FACS-coupled RNA-seq compared with alternative gene-expression methodologies, including quantitative PCR (qPCR), bulk root RNA-seq, single-cell RNA-seq (scRNA-seq), and spatial transcriptomics, considering sensitivity, resolution, input requirements, and cost.