Comprehensive Spatial Profiling of Species-agnostic Transcriptomes <em>via</em> Stereo-seq

作者：Tod D. Casasent, Danielle L. Stolley, Basant T. Gamal, Arnold Dahay, Samuel C. Mok, Lidia Rocha, Vincent Li, Arizona T. Nguyen, Boyu Zhang, Clay T. Brasuell, Erika J. Thompson, Thomas V. Huynh, Jared K. Burks, Sammy Ferri‐Borgogno · 发表于：Journal of Visualized Experiments · 年份：2025 · DOI：10.3791/68619 · 研究领域：Single-cell and spatial transcriptomics、Molecular Biology Techniques and Applications、Gene expression and cancer classification

The spatial composition of cells within the host, as well as the bacteria or viral loads within the tissue, can impact the interaction of cell types and analytes that drive the cell through cell-type-specific processes. Stereo-seq for FFPE tissues uses random priming to spatial barcodes, which is different from standard spatial transcriptomics methods, which use A'tailing to capture messenger RNA (mRNA) or probe-based to capture species-specific transcripts. These methods do not embrace the more current knowledge about the impact and importance of other types of RNA from long non-coding RNA, mitochondrial RNA, microRNA, or other species, RNA, such as microbial, viral, and fungal. Outlined here is a step-by-step procedure from tissue sectioning through library preparation for spatial species-agnostic stereo-seq application with RNA detection using a randomer probe methodology, which is compatible with formalin fixed paraffin embedded (FFPE) tissues. This Stereo-seq method has a random capture bead of 0.22 µm in size that reoccurs in an array every 0.5 µm, allowing for subcellular resolution from a sequencing-based technology. As this method detects both host and non-host RNA, the protocol requires specific considerations to allow for the determination of what is inside the tissue versus what was deposited on the tissue during the collection, preservation, cutting, and detection process (i.e., environmental and handling contaminants). Lastly, this protocol allows one to have hi...