Abstract 4088: Optimized workflow for isolating nuclei from FFPE tissue sections for snRNAseq

Background: Formaldehyde fixed and paraffin embedded (FFPE) tissue sections are a valuable resource in cancer research due to the long-term preservation of clinical samples. However, these tissues present significant challenges for molecular analysis due to fixation-induced crosslinking. This study describes an optimized workflow that enables efficient isolation of nuclei from FFPE tissues, compatible with the Chromium Single Cell Gene Expression Flex Assay for downstream gene expression analysis. Methods: FFPE tissue sections from different tissue types (i.e., breast-, lung-, and colon-cancer) were subjected to a deparaffinization and rehydration process, followed by a combined gentleMACS based mechanical dissociation and enzymatic digestion to obtain nuclei. Our new dissociation protocol was benchmarked against a Liberase TH based dissociation. Key modifications of our protocol include the selection of an optimized enzyme mix and a digestion buffer composition to enhance nuclei yield and purity. Nuclei purity and yield were quantified using flow cytometry and an automated cell counter, respectively, while RNA metrics, such as DV200, were assessed via the Agilent TapeStation. The isolated nuclei were then processed using 10x Genomics Chromium Single Cell Gene Expression Flex Assay to validate their suitability for RNA profiling. Results: The optimized protocol successfully isolated nuclei from a variety of FFPE tissue types, often yielding sufficient nuclei from a single 25um FFPE tissue scroll. The following Chromium Single Cell Gene Expression Flex Assay confirmed the superior performance of our new dissociation workflow for RNA profiling. Cell Rangers QC metrics for median UMI counts, median genes, and median reads per cell were improved on average by 61%, 53%, and 59%, respectively. Furthermore, mitochondrial gene expression was reduced on average by 63% compared to the control, suggesting a cleaner nuclei sample. Conclusion: The optimized workflow provides a significant advancement for utilizing FFPE samples in single-cell gene expression studies. By enabling efficient nuclei isolation from FFPE sections, this protocol allows for comprehensive transcriptomic analysis using the Chromium Single Cell Gene Expression Flex Assay, with minimal background, unlocking the potential of archived tissues in retrospective and translational research. Citation Format: Lea Eich, Svetlana Khorkova, Nojan Jelveh, Lilian Martinez Carrera, Olaf Hardt, Carsten Poggel, Benjamin Theek. Optimized workflow for isolating nuclei from FFPE tissue sections for snRNAseq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4088.