A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome

Traditional tissue dissociation methods for bulk- and single-cell sequencing use various protease and/or collagenase combinations at temperatures ranging from 28 to 37 °C, which cause transcriptional cell stress that may alter data interpretation. Such artifacts can be reduced by dissociating cells in cold-active proteases, but few studies have shown that this improves cell-type specific transcription, particularly in tissues hypersensitive to mechanical integrity and extracellular matrix (ECM) interactions. To address this, we have dissociated zebrafish tendons and ligaments in subtilisin A at 4 °C and compared the results with 37 °C collagenase dissociation using bulk RNA sequencing. We find that high-temperature collagenase dissociation causes general cell stress in tendon fibroblasts (tenocytes) as reported in previous studies with other cell types, but also that high temperature specifically downregulates hallmark genes involved in tenocyte specification and ECM production in vivo. Our results suggest that cold-protease dissociation reduces transcriptional artifacts and increases the robustness of RNA-sequencing datasets such that they better reflect native in vivo tissue microenvironments. Key features • Utilizing a cold-active protease derived from the Himalayan soil bacterium <i>B. licheniformis</i> for tissue dissociation preserves cell transcriptomes, increasing data quality of downstream sequencing experiments. • This method is reproducible and requires no extra equipment for tissue agitation. • Tenocytes isolated using this method show lower stress and better preserved native expression of key tenocyte markers and ECM genes than with traditional warm-dissociation methods. • This protocol is ideal for cell types that are particularly sensitive to microenvironment signals or are embedded in extracellular matrix.