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

Formalin-fixed paraffin-embedded (FFPE) samples remain an underutilized resource in single-cell omics due to RNA degradation from formalin fixation. Here, we present snPATHO-seq, a robust and adaptable approach that enables the generation of high-quality single-nucleus (sn) transcriptomic data from FFPE tissues, utilizing advancements in single-cell genomic techniques. The snPATHO-seq workflow integrates optimized nuclei isolation with the 10× Genomics Flex assay, targeting short RNA fragments to mitigate FFPE-related RNA degradation. Benchmarking against standard 10× 3' and Flex assays for fresh/frozen tissues confirmed robust detection of transcriptomic signatures and cell types. snPATHO-seq demonstrated high performance across diverse FFPE samples, including diseased tissues like breast cancer. It seamlessly integrates with FFPE spatial transcriptomics (e.g., FFPE Visium) for multi-modal spatial and single-nucleus profiling. Compared to workflows like 10× Genomics' snFFPE, snPATHO-seq delivers superior data quality by reducing tissue debris and preserving RNA integrity via nuclei isolation. This cost-effective workflow enables high-resolution transcriptomics of archival FFPE samples, advancing single-cell omics in translational and clinical research. Key features • Optimized nuclei isolation from FFPE tissues enables high-quality single-nucleus transcriptomics by minimizing debris and maximizing intact nuclear yield. • Compatible with 10× Genomics Flex, leveraging short RNA probes to overcome FFPE RNA fragmentation challenges. • Outperforms existing FFPE workflows in cell type detection sensitivity across archival, degraded, or aged samples. • Low-cost, accessible protocol using off-the-shelf reagents, suitable for broad translational and archival tissue applications.