Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy

Abstract

PurposeProliferative 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. Here, we aimed to compare single-cell and single-nucleus RNA sequencing (scRNA-Seq or snRNA-Seq) of retinal PVR samples in the rabbit model.MethodsPVR was induced unilaterally in Dutch Belted rabbits. At different timepoints following PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for scRNA-Seq or snRNA-Seq .ResultsScRNA-Seq and snRNA-Seq were conducted on retinas at 4 hours and 14 days after disease induction. While the capture rate of unique molecular identifiers (UMI) 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 two sequencing modalities is the cell type capture rate, however, with glial cell types over-represented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Müller glia were over-represented in snRNA-seq samples, while reactive Müller glia were in scRNA-seq samples. Trajectory analyses were similar between the two methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq datasets.ConclusionsThese 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. 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. Here, we aimed to compare single-cell and single-nucleus RNA sequencing (scRNA-Seq or snRNA-Seq) of retinal PVR samples in the rabbit model. PVR was induced unilaterally in Dutch Belted rabbits. At different timepoints following PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for scRNA-Seq or snRNA-Seq . ScRNA-Seq and snRNA-Seq were conducted on retinas at 4 hours and 14 days after disease induction. While the capture rate of unique molecular identifiers (UMI) 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 two sequencing modalities is the cell type capture rate, however, with glial cell types over-represented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Müller glia were over-represented in snRNA-seq samples, while reactive Müller glia were in scRNA-seq samples. Trajectory analyses were similar between the two methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq datasets. 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.

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