Protocol for isolating high-quality nuclei from FFPE pancreatic cancer sections and constructing sequencing libraries.

Formalin-fixed paraffin-embedded (FFPE) tissues are archived resources for biomedical research but challenging for single-cell transcriptomic analysis. Here, we present a protocol for isolating high-quality nuclei from FFPE pancreatic cancer sections. We describe steps for deparaffinization, enzymatic digestion, nuclei purification, and library construction using the 10× Genomics Fixed RNA Profiling platform. This protocol enables transcriptomic profiling of archived clinical specimens at single-nucleus resolution. For complete details on the use and execution of this protocol, please refer to Chen et al.1.