Single-cell RNA-seq of Retinal Cells

Single-cell RNA sequencing (scRNA)-seq analysis was carried out as previously described (Xiao et al., 2020 (link)). In brief, 5.5 days after infection of adult mouse retinas with GFAP-Math5-Brn3b-GFP AAVs, two retinas were quickly dissected and dissociated using papain with DNase I at 37°C for 5 min. Then isometric amount of DPBS containing 10% FBS was added and retinas were triturated by soft pipetting for dissociation. The dissociated cells were filtered using a 40-μm cell strainer. Filtered cells were centrifuged and resuspended with 2 ml DPBS containing 5% FBS. GFP⁺ retinal cells were then enriched by fluorescence-activated cell sorting (FACS) using the FACSAria Fusion cell sorter (BD Biosciences). Single-cell libraries were generated from the enriched GFP+ cells and sequenced on the Illumina X Ten platform (Berry Genomics, China). Further analyses were performed using Seurat and Monocle (Xiao et al., 2020 (link)).