snPATHO-seq: A Detailed Protocol for Single Nucleus RNA Sequencing From FFPE.

Area of Science:

• Single-cell genomics
• Molecular pathology
• Biotechnology

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource but challenging for single-cell omics due to RNA degradation.
• Existing methods struggle to yield high-quality transcriptomic data from FFPE samples, limiting their use in research.

Purpose of the Study:

• To present snPATHO-seq, a novel workflow for generating high-quality single-nucleus (sn) transcriptomic data from FFPE tissues.
• To overcome limitations of RNA degradation in FFPE samples for advanced single-cell omics analysis.

Main Methods:

• Developed snPATHO-seq integrating optimized nuclei isolation from FFPE tissues with the 10× Genomics Flex assay.
• Targeted short RNA fragments to mitigate FFPE-related RNA degradation.
• Benchmarked against standard assays on fresh/frozen and diverse FFPE tissues, including breast cancer samples.

Main Results:

• snPATHO-seq demonstrated robust detection of transcriptomic signatures and cell types across various FFPE samples.
• Achieved superior data quality compared to existing FFPE workflows, reducing tissue debris and preserving RNA integrity.
• Showcased high performance and cell type detection sensitivity, outperforming other FFPE methods.

Conclusions:

• snPATHO-seq provides a cost-effective and adaptable solution for high-resolution transcriptomics of archival FFPE samples.
• This workflow advances single-cell omics applications in translational and clinical research by unlocking the potential of FFPE tissues.
• Enables multi-modal spatial and single-nucleus profiling when integrated with FFPE spatial transcriptomics.