Formalin-fixed, paraffin-embedded (FFPE) tissues represent a vast and underutilized resource of patient data for retrospective and translational research. While FFPE enables long-term preservation of valuable clinical specimens, RNA degradation and fragmentation have historically hindered its compatibility with single cell RNA sequencing (scRNA-seq). Currently, researchers have relied on probe-based FFPE profiling approaches and are limited to predefined target sets, restricting transcriptome coverage and discovery potential.
Here, we introduce a novel split-pool combinatorial barcoding method for FFPE tissue that captures the full transcriptome through dual priming with both poly dT and random hexamer reverse transcription primers. This workflow extends the scalability of Parse’s single-cell technology to FFPE tissues, enabling unbiased transcriptome-wide analysis of millions of cells and hundreds of samples within a single experiment. By capturing a broad range of RNA biotypes, this approach delivers deeper insights into cellular function and disease mechanisms.
As a proof of concept, FFPE tissues from both human and mouse were processed. The resulting single cell datasets demonstrated high-resolution identification of cell types across diverse tissue types. Comparative analyses between FFPE and matched fresh frozen samples revealed concordance in gene expression patterns and cell type proportions, validating the approach’s complete capture of sample biology.
