Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq.

Area of Science:

• Spatial transcriptomics
• Molecular biology
• Genomics

Background:

• Cellular spatial composition and microbial loads influence host-tissue interactions.
• Existing spatial transcriptomics methods often miss non-messenger RNA (mRNA) species like long non-coding RNA, mitochondrial RNA, and microbial RNA.
• Formalin-fixed paraffin-embedded (FFPE) tissues are valuable but challenging for comprehensive spatial RNA analysis.

Purpose of the Study:

• To present a detailed protocol for spatial species-agnostic Stereo-seq on FFPE tissues.
• To enable detection of a wide range of RNA species, including host and non-host RNA, with subcellular resolution.
• To provide insights into the spatial distribution of diverse RNA molecules within tissues.

Main Methods:

• Utilizes a random priming approach with spatial barcodes for RNA capture in FFPE tissues.
• Employs a randomer probe methodology for species-agnostic RNA detection.
• Incorporates specific considerations for distinguishing endogenous RNA from environmental contaminants during sample processing.

Main Results:

• Achieves subcellular resolution (0.22 µm beads, 0.5 µm array) for spatial RNA detection.
• Successfully detects both host and non-host RNA (microbial, viral) within FFPE tissues.
• Provides high-resolution spatial mapping of multiple RNA species, including single-cell level resolution.

Conclusions:

• The developed spatial Stereo-seq protocol is compatible with FFPE tissues and offers broad RNA species detection.
• This method enhances understanding of host-microbe and host-pathogen interactions in a spatial context.
• It offers significant potential for studying complex tissue microenvironments and disease pathology.