A two-stage digestion of whole murine knee joints for single-cell RNA sequencing
Objective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | Osteoarthritis and Cartilage Open |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2665913122000899 |
| _version_ | 1797991809957232640 |
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| author | Dustin M. Leale Linan Li Matthew L. Settles Keith Mitchell Lutz Froenicke Jasper H.N. Yik Dominik R. Haudenschild |
| author_facet | Dustin M. Leale Linan Li Matthew L. Settles Keith Mitchell Lutz Froenicke Jasper H.N. Yik Dominik R. Haudenschild |
| author_sort | Dustin M. Leale |
| collection | DOAJ |
| description | Objective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties. Design: A two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform. Results: We observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types. Conclusions: The knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ. |
| first_indexed | 2024-04-11T08:58:05Z |
| format | Article |
| id | doaj.art-2a8af5a621134bfcb781f0496b0e694f |
| institution | Directory Open Access Journal |
| issn | 2665-9131 |
| language | English |
| last_indexed | 2024-04-11T08:58:05Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Osteoarthritis and Cartilage Open |
| spelling | doaj.art-2a8af5a621134bfcb781f0496b0e694f2022-12-22T04:33:05ZengElsevierOsteoarthritis and Cartilage Open2665-91312022-12-0144100321A two-stage digestion of whole murine knee joints for single-cell RNA sequencingDustin M. Leale0Linan Li1Matthew L. Settles2Keith Mitchell3Lutz Froenicke4Jasper H.N. Yik5Dominik R. Haudenschild6Department of Orthopaedic Surgery, UC Davis School of Medicine, Sacramento, CA, USA; Corresponding author. 2700 Stockton Blvd, Suite 2407, Sacramento, CA, 95817, USA.Department of Orthopaedic Surgery, UC Davis School of Medicine, Sacramento, CA, USAUC Davis Bioinformatics Core Facility, Davis, CA, USAUC Davis Bioinformatics Core Facility, Davis, CA, USAUC Davis DNA Technologies and Expression Analysis Cores, Davis, CA, USADepartment of Orthopaedic Surgery, UC Davis School of Medicine, Sacramento, CA, USADepartment of Orthopaedic Surgery, UC Davis School of Medicine, Sacramento, CA, USAObjective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties. Design: A two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform. Results: We observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types. Conclusions: The knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ.http://www.sciencedirect.com/science/article/pii/S2665913122000899Single-cell RNA sequencingOsteoarthritisWhole knee jointTissue cross-talkMouse model |
| spellingShingle | Dustin M. Leale Linan Li Matthew L. Settles Keith Mitchell Lutz Froenicke Jasper H.N. Yik Dominik R. Haudenschild A two-stage digestion of whole murine knee joints for single-cell RNA sequencing Osteoarthritis and Cartilage Open Single-cell RNA sequencing Osteoarthritis Whole knee joint Tissue cross-talk Mouse model |
| title | A two-stage digestion of whole murine knee joints for single-cell RNA sequencing |
| title_full | A two-stage digestion of whole murine knee joints for single-cell RNA sequencing |
| title_fullStr | A two-stage digestion of whole murine knee joints for single-cell RNA sequencing |
| title_full_unstemmed | A two-stage digestion of whole murine knee joints for single-cell RNA sequencing |
| title_short | A two-stage digestion of whole murine knee joints for single-cell RNA sequencing |
| title_sort | two stage digestion of whole murine knee joints for single cell rna sequencing |
| topic | Single-cell RNA sequencing Osteoarthritis Whole knee joint Tissue cross-talk Mouse model |
| url | http://www.sciencedirect.com/science/article/pii/S2665913122000899 |
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