Single-Cell Library Generation from Retina

Single cell libraries were generated by minor modifications of methods developed for macaque retina^{12 (link)}. Briefly, a ~1.5 mm diameter circular region centered on the foveal pit was dissected from the retina, and peripheral retinal pieces were pooled from all retinal quadrants. Dissected tissues were digested with papain (Worthington, LS003126) for 30 min at 37 °C. Following digestion, samples were dissociated and triturated into single cell suspensions with 0.04% bovine serum albumin (BSA) in Ames. Dissociated cells from digested peripheral retinas were incubated with CD90 microbeads (Miltenyi Biotec, 130-096-253; 1 ml per 10⁷ cells) to enrich RGCs or with anti-CD73 (BD Biosciences, clone AD2; 5 ml per 10⁷ cells) followed by anti-mouse IgG1 microbeads (Miltenyi Biotec, 130-047-102; 10 ml per 10⁷ cells) to deplete rods. Incubations were at room temperature for 10 min. CD90 positive cells or CD73 negative cells were selected via large cell columns through a MiniMACS Separator (Miltenyi Biotec). Foveal samples were used without further processing. Single cell suspensions were diluted to 500–1800 cells/µL in 0.04% BSA/Ames for loading into 10X Chromium Single Cell v2 or v3 Chips. Following collection, cDNA libraries were prepared following the manufacturer’s protocol, and sequenced on the Illumina HiSeq. 2500 (Paired end reads: Read 1, 26 bp, Read 2, 98 bp).