Thomson Benjamin R, Quaggin Susan E
Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Department of Ophthalmology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Bio Protoc. 2022 May 20;12(10). doi: 10.21769/BioProtoc.4426.
Single cell RNA sequencing is a powerful tool that can be used to identify distinct cell types and transcriptomic differences within complex tissues. It has proven to be especially useful in tissues of the eye, where investigators have identified novel cell types within the retina, anterior chamber, and iridocorneal angle and explored transcriptomic contribution to disease phenotypes in age-related macular degeneration. However, to obtain high quality results, the technique requires isolation of healthy single cells from the tissue of interest, seeking complete tissue digestion while minimizing stress and transcriptomic changes in the isolated cells prior to library preparation. Here, we present a protocol developed in our laboratory for isolation of live single cells from the murine iridocorneal angle, which includes Schlemm's canal and the trabecular meshwork, suitable for single cell RNA sequencing, flow cytometry, or other downstream analysis. Graphical abstract.
单细胞RNA测序是一种强大的工具，可用于识别复杂组织内不同的细胞类型和转录组差异。事实证明，它在眼部组织中特别有用，研究人员在那里识别出视网膜、前房和虹膜角膜角内的新型细胞类型，并探索了转录组对年龄相关性黄斑变性疾病表型的影响。然而，为了获得高质量的结果，该技术需要从感兴趣的组织中分离出健康的单细胞，在文库制备之前寻求完全的组织消化，同时尽量减少分离细胞中的应激和转录组变化。在这里，我们展示了我们实验室开发的一种从鼠虹膜角膜角分离活单细胞的方案，该方案包括施莱姆管和小梁网，适用于单细胞RNA测序、流式细胞术或其他下游分析。图形摘要。
