Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy
Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery, and the molecular changes leading to this aberrant wound healing process are currently unknown. Our ultimate goal is to study PVR pathogenesis by employing single-cell transcriptomics...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-12-01
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| Series: | Ophthalmology Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666914523000672 |
| _version_ | 1827400226109915136 |
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| author | Clayton P. Santiago, PhD, MS Megan Y. Gimmen, BSc Yuchen Lu, BSc Minda M. McNally, MS Leighton H. Duncan, MA, BSc Tyler J. Creamer, PhD Linda D. Orzolek, MS Seth Blackshaw, PhD Mandeep S. Singh, MD, PhD |
| author_facet | Clayton P. Santiago, PhD, MS Megan Y. Gimmen, BSc Yuchen Lu, BSc Minda M. McNally, MS Leighton H. Duncan, MA, BSc Tyler J. Creamer, PhD Linda D. Orzolek, MS Seth Blackshaw, PhD Mandeep S. Singh, MD, PhD |
| author_sort | Clayton P. Santiago, PhD, MS |
| collection | DOAJ |
| description | Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery, and the molecular changes leading to this aberrant wound healing process are currently unknown. Our ultimate goal is to study PVR pathogenesis by employing single-cell transcriptomics to dissect cellular heterogeneity. Design: Here we aimed to compare single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-sequencing (snRNA-seq) of retinal PVR samples in the rabbit model. Participants: Unilateral induction of PVR lesions in rabbit eyes with contralateral eyes serving as controls. Methods: Proliferative vitreoretinopathy was induced unilaterally in Dutch Belted rabbits. At different timepoints after PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for scRNA-seq or snRNA-seq. Main Outcome Measures: Single cell and nuclei transcriptomic profiles of retinas after PVR induction. Results: Single-cell RNA sequencing and snRNA-seq were conducted on retinas at 4 hours and 14 days after disease induction. Although the capture rate of unique molecular identifiers and genes were greater in scRNA-seq samples, overall gene expression profiles of individual cell types were highly correlated between scRNA-seq and snRNA-seq. A major disparity between the 2 sequencing modalities was the cell type capture rate, however, with glial cell types overrepresented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Müller glia were overrepresented in snRNA-seq samples, whereas reactive Müller glia were overrepresented in scRNA-seq samples. Trajectory analyses were similar between the 2 methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq data sets. Conclusions: These findings highlight limitations of both scRNA-seq and snRNA-seq analysis and imply that use of both techniques together can more accurately identify transcriptional networks critical for aberrant fibrogenesis in PVR than using either in isolation. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article. |
| first_indexed | 2024-03-08T19:57:55Z |
| format | Article |
| id | doaj.art-33c3d0e044e2446db6f33df651349f50 |
| institution | Directory Open Access Journal |
| issn | 2666-9145 |
| language | English |
| last_indexed | 2024-03-08T19:57:55Z |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Ophthalmology Science |
| spelling | doaj.art-33c3d0e044e2446db6f33df651349f502023-12-24T04:47:05ZengElsevierOphthalmology Science2666-91452023-12-0134100335Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative VitreoretinopathyClayton P. Santiago, PhD, MS0Megan Y. Gimmen, BSc1Yuchen Lu, BSc2Minda M. McNally, MS3Leighton H. Duncan, MA, BSc4Tyler J. Creamer, PhD5Linda D. Orzolek, MS6Seth Blackshaw, PhD7Mandeep S. Singh, MD, PhD8Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MarylandSolomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MarylandWilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MarylandWilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MarylandSolomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MarylandInstitute for Basic Biomedical Sciences, Johns Hopkins University, Baltimore, MarylandInstitute for Basic Biomedical Sciences, Johns Hopkins University, Baltimore, MarylandSolomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland; Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland; Kavli Neuroscience Discovery Institute, Johns Hopkins University, Baltimore, Maryland; Seth Blackshaw, PhD, MRB 339, 733 N. Broadway Avenue, Baltimore, MD 21205.Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; Correspondence: Mandeep S. Singh, MD, PhD, 600 N. Wolfe Street, Baltimore MD 21287.Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery, and the molecular changes leading to this aberrant wound healing process are currently unknown. Our ultimate goal is to study PVR pathogenesis by employing single-cell transcriptomics to dissect cellular heterogeneity. Design: Here we aimed to compare single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-sequencing (snRNA-seq) of retinal PVR samples in the rabbit model. Participants: Unilateral induction of PVR lesions in rabbit eyes with contralateral eyes serving as controls. Methods: Proliferative vitreoretinopathy was induced unilaterally in Dutch Belted rabbits. At different timepoints after PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for scRNA-seq or snRNA-seq. Main Outcome Measures: Single cell and nuclei transcriptomic profiles of retinas after PVR induction. Results: Single-cell RNA sequencing and snRNA-seq were conducted on retinas at 4 hours and 14 days after disease induction. Although the capture rate of unique molecular identifiers and genes were greater in scRNA-seq samples, overall gene expression profiles of individual cell types were highly correlated between scRNA-seq and snRNA-seq. A major disparity between the 2 sequencing modalities was the cell type capture rate, however, with glial cell types overrepresented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Müller glia were overrepresented in snRNA-seq samples, whereas reactive Müller glia were overrepresented in scRNA-seq samples. Trajectory analyses were similar between the 2 methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq data sets. Conclusions: These findings highlight limitations of both scRNA-seq and snRNA-seq analysis and imply that use of both techniques together can more accurately identify transcriptional networks critical for aberrant fibrogenesis in PVR than using either in isolation. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.http://www.sciencedirect.com/science/article/pii/S2666914523000672Single cell RNA-sequencingSingle nucleus RNA-sequencingProliferative vitreoretinopathyFibrosis |
| spellingShingle | Clayton P. Santiago, PhD, MS Megan Y. Gimmen, BSc Yuchen Lu, BSc Minda M. McNally, MS Leighton H. Duncan, MA, BSc Tyler J. Creamer, PhD Linda D. Orzolek, MS Seth Blackshaw, PhD Mandeep S. Singh, MD, PhD Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy Ophthalmology Science Single cell RNA-sequencing Single nucleus RNA-sequencing Proliferative vitreoretinopathy Fibrosis |
| title | Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy |
| title_full | Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy |
| title_fullStr | Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy |
| title_full_unstemmed | Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy |
| title_short | Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy |
| title_sort | comparative analysis of single cell and single nucleus rna sequencing in a rabbit model of retinal detachment related proliferative vitreoretinopathy |
| topic | Single cell RNA-sequencing Single nucleus RNA-sequencing Proliferative vitreoretinopathy Fibrosis |
| url | http://www.sciencedirect.com/science/article/pii/S2666914523000672 |
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