Automated in situ microfluidic Random-seq for robust single-nucleus and spatial total RNA profiling of diverse FFPE specimens.

Formalin-fixed paraffin-embedded (FFPE) tissues are indispensable for clinical- pathological assessment, yet widespread nucleic acid degradation severely hinders high-throughput molecular profiling. Current RNA sequencing and in situ hybridization approaches for FFPE materials suffer from inconsistent performance across tissue types and preservation conditions, restricting standardized, automated transcriptome analysis. Here, we show an in situ microfluidic Random-seq (imRandom-seq) platform that enables unified bulk, single-nucleus, and spatial total RNA profiling of FFPE specimens. Using specially designed random primers for efficient transcript capture and total transcriptome analysis, single-nucleus imRandom-seq outperforms traditional snRNA-seq and probe-based 10X Flex, with enhanced gene detection, reduced nuclear loss, and biologically reasonable cell-type annotation. Validated in difficult samples with high enzymatic activity and fragmented RNA, this in situ microfluidics-driven workflow features low manual operation, broad tissue compatibility, and robust data quality, providing a reliable and scalable tool for FFPE transcriptomic research.