Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland.
Genomics Core Technology Unit, Faculty of Medicine, Health & Life Sciences, Queen's University Belfast, Belfast, Northern Ireland.
Mol Vis. 2020 Nov 27;26:766-779. eCollection 2020.
To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs).
Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses.
Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively.
We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.
通过分析原代牛视网膜微血管内皮细胞（RMEC）的单个转录组，更好地描述视网膜内皮屏障特性。
单个 RMEC 在 Fluidigm C1 系统上捕获，使用 Nextera XT 试剂盒制备 cDNA 文库，并在 NextSeq 系统（Illumina）上进行测序。使用 R 包 Scater、SC3 和 Seurat 以及浏览器应用程序 Automated Single-cell Analysis Pipeline（ASAP）进行数据分析。单细胞中的选择性剪接事件用 Outrigger 进行定量。使用 Cytoscape 进行网络分析。
应用单细胞 RNA 测序（scRNA-seq）分析工作流程表明，RMEC 在培养中形成相对同质的群体，主要差异与增殖状态有关。沿动静脉树表达的标志物表明，大多数细胞源自毛细血管。所有细胞的平均基因表达水平用于开发内在血视网膜屏障的计算机模型，该模型纳入了以前未在视网膜血管中报道的连接蛋白。对单个细胞中屏障基因表达的相关性分析揭示了一个亚组基因与相关性网络中心的 PECAM-1 高度相关。观察到涉及微血管屏障基因内外显子的大量选择性剪接事件，在许多情况下，单个细胞仅表达一种同工型。
我们优化了原代 RMEC 单细胞转录组学的工作流程。结果为参与视网膜微血管屏障形成的基因提供了基本的见解。
