snPATHO-seq, a versatile FFPE single-nucleus RNA sequencing method to unlock pathology archives

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are a rich source of clinical samples but are challenging for single-cell omics due to RNA degradation.
• Existing single-cell RNA sequencing (scRNA-seq) methods are often suboptimal for FFPE samples, limiting their utility.

Purpose of the Study:

• To introduce and validate snPATHO-seq, a novel method for generating high-quality single-nucleus transcriptomic data from FFPE samples.
• To benchmark snPATHO-seq against established scRNA-seq assays for fresh or frozen tissues.
• To demonstrate the robustness and applicability of snPATHO-seq across diverse FFPE tissue types.

Main Methods:

• Development and implementation of the snPATHO-seq workflow for FFPE sample processing.
• Comparative analysis of snPATHO-seq data with 10x Genomics 3' and Flex assays.
• Testing snPATHO-seq on a variety of healthy and diseased FFPE tissue samples.
• Integration of snPATHO-seq with FFPE spatial transcriptomic technologies.

Main Results:

• snPATHO-seq successfully generates high-quality single-nucleus transcriptomic data from FFPE samples.
• The method shows consistency with established scRNA-seq workflows (10x 3' and Flex).
• The snPATHO-seq workflow is robust across a wide spectrum of FFPE tissue types.
• snPATHO-seq enables multi-modal analysis when combined with spatial transcriptomics.

Conclusions:

• snPATHO-seq significantly enhances the utility of FFPE samples for single-nucleus transcriptomic studies.
• This method overcomes previous limitations associated with RNA quality in FFPE tissues.
• snPATHO-seq offers a versatile approach for comprehensive transcriptomic characterization of FFPE samples, alone or in combination with spatial data.