Enrichment of Retinal Endothelial Cells and Müller Glia Enable Comprehensive scRNA-Seq Profiling of the Blood-Retinal Barrier.

Obtaining high-quality single-cell suspensions from neural tissues such as the murine retina remains technically challenging due to cellular fragility, tight intercellular junctions, and the heterogeneous composition of the tissue. These challenges are further compounded when studying rare cell populations, including vascular endothelial cells and Müller glia, which each account for less than 2% and are underrepresented in standard dissociation workflows, often lost during processing. Here, we present an optimized method for isolating murine retinal cells that preserves cellular integrity and enables selective enrichment of sparse populations for downstream single-cell RNA sequencing (scRNA-seq). Our protocol integrates gentle enzymatic dissociation with mechanical trituration to maximize cell viability and minimize transcriptional artifacts. We further incorporate targeted enrichment strategies, using fluorescence-activated cell sorting (FACS), to increase the relative abundance of vascular endothelial and Müller glial cells without compromising overall transcriptomic diversity. Moreover, we tested antibody-based cell hashing to enable sample multiplexing to improve cost efficiency. We demonstrate that this approach yields high-quality single-cell suspensions with improved representation of rare retinal cell types, enabling more robust and reproducible scRNA-seq analyses. This method provides a practical framework for researchers aiming to investigate the vascular and glial cell components of the retina at single-cell resolution and can be adapted to other neural tissues in which rare cell populations are of interest.