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...
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Bio-protocol LLC
2025-05-01
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| author | Arul Subramanian Pavan Nayak Thomas Schilling |
| author_facet | Arul Subramanian Pavan Nayak Thomas Schilling |
| author_sort | Arul Subramanian |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-16efbd256c614bf4b9c71e5042287e3a |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-16efbd256c614bf4b9c71e5042287e3a2025-08-20T02:14:01ZengBio-protocol LLCBio-Protocol2331-83252025-05-0115910.21769/BioProtoc.5293A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast TranscriptomeArul Subramanian0Pavan Nayak1Thomas Schilling2Department of Developmental and Cell Biology, University of California, Irvine, CA, USADepartment of Developmental and Cell Biology, University of California, Irvine, CA, USADepartment of Developmental and Cell Biology, University of California, Irvine, CA, USATraditional 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.https://bio-protocol.org/en/bpdetail?id=5293&type=0 |
| spellingShingle | Arul Subramanian Pavan Nayak Thomas Schilling A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome Bio-Protocol |
| title | A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome |
| title_full | A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome |
| title_fullStr | A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome |
| title_full_unstemmed | A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome |
| title_short | A Cold-Active Protease Tissue Dissociation Protocol for the Preservation of the Tendon Fibroblast Transcriptome |
| title_sort | cold active protease tissue dissociation protocol for the preservation of the tendon fibroblast transcriptome |
| url | https://bio-protocol.org/en/bpdetail?id=5293&type=0 |
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