A practical guide to spatial transcriptomics: lessons from over 1000 samples

Spatial transcriptomics (ST) enables the  in situ mapping of gene expression, revolutionizing our ability to study tissue organization and cellular interactions. However, many groups struggle with practical barriers to implementation, including platform selection, sample quality, and experimental scalability. We provide a practical guide to ST, informed by the processing and analysis of over 1000 spatial samples across multiple ST platforms. We outline best practices for experimental design, tissue handling, sequencing, and computational analysis, with special attention to clinical samples. Our goal is to translate hands-on experience into 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 large-scale studies.