Sample Preparation for Imaging-Based Spatial Transcriptomics in Rigid Plant Tissues (Roots, Shoots).

Plant roots dynamically respond to environmental changes and serve as an ideal system for studying cell development and gene regulation. Recent advances in imaging-based spatial transcriptomics have enabled high-resolution mapping of gene expression while preserving spatial context. However, existing sample preparation techniques remain inadequate for handling rigid plant tissues such as crop roots. Here, we present a detailed and practical protocol for preparing rigid plant tissue samples for imaging-based spatial transcriptomics. The workflow ensures effective tissue handling while maintaining RNA integrity and spatial organization. Within approximately eight days, samples can be processed and mounted onto commercial slides, making them ready for subsequent probe hybridization and imaging. This protocol also includes an integrated sample attachment test performed to assess slide quality. It has been optimized to produce consistent and reliable results across experiments. Overall, our method provides a robust solution for spatial transcriptomic analysis in rigid plant tissues, facilitating broader application of these technologies in plant research. Key features • Builds upon the method developed by Zhu et al. [1] and introduces an optimized sample preparation protocol for imaging-based spatial transcriptomics in rigid rice roots. • Ensures effective tissue fixation and sectioning, while preserving RNA integrity and spatial organization. • Includes an integrated sample attachment test to assess the adhesion of tissue sections to commercial slides. • Requires approximately 8 days to complete the sample preparation, with another 6 days for the attachment test.