A Practical Guide to Spatial Transcriptomics: Lessons from over 1000 Samples

Spatial transcriptomics enables the in situ mapping of gene expression, revolutionizing our ability to study tissue organization and cellular interactions. However, as this technology is increasingly adopted across biological and clinical research, many groups struggle with practical barriers to implementation—including platform selection, sample quality, and experimental scalability.Here, we provide a comprehensive, practical guide to spatial transcriptomics, informed by the processing and analysis of over 1,000 spatial samples across Visium, Visium HD, and Xenium platforms. We outline best practices for experimental design, tissue handling, sequencing, and computational analysis, with special attention to clinical samples and high-throughput settings.Our goal is to translate hands-on experience into actionable recommendations that support robust, reproducible spatial workflows. This guide is designed to assist researchers at all levels—from those designing their first spatial experiment to groups aiming to integrate ST into translational pipelines and large-scale studies.