Non acetylated bovine serum albumin

Ten representative organoids were selected from culture. Organoids were allowed to settle by gravity in a 1.5 mL tube and medium was aspirated. Organoids were then dissociated with 20 units / mL of papain (Worthington Biochemical Corporation, Lakewood, NJ) with 60 units / mL DNase I (Worthington Biochemical Corporation) on a shaker at 37 ˚C for approximately 60 min. Following dissociation, cells were pelleted at 400 × g for 5 min, resuspended in dPBS -/- (Thermo Fisher Scientific) with 0.04% non-acetylated bovine serum albumin (New England Biolabs, Ipswich, MA), and processed immediately for encapsulation and barcoding using the Chromium Controller (10X Genomics, Pleasanton, CA).

Retinal tissue was isolated from donors 5–8 with 1 mm (foveal) and 4 mm (parafoveal) trephine punch biopsies centered on the fovea. To minimize inadvertently collecting cells from the retinal pigment epithelium or choroid, an 8 mm retinal punch was first collected and moved to a separate dish, and the 1 and 4 mm samples were collected from the excised macula before gentle dissociation in 20 units/ml of papain with 0.005% DNase I (Worthington Biochemical Corporation, Lakewood NJ, USA) on a shaker at 37°C for 1–1.25 h. The dissociated retinal samples were resuspended in DMSO-based Recovery Cell Culture Freezing Media (Life Technologies, Grand Island NY, USA) and frozen at −80°C in a CoolCell LX container (Corning NY, USA) to cool at 1°C/min. After samples were frozen for 3–12 h, they were transferred to liquid nitrogen for long-term storage.
Cryopreserved retinal samples were rapidly thawed in parallel at 37°C and resuspended in phosphatidylcholine buffer solution with 0.04% non-acetylated bovine serum albumin (New England Biolabs, Ipswich MA, USA) at a concentration of 1000 cells/μl. Single cells were then microfluidically separated and barcoded with the Chromium system v3.1 chemistry kit (10X Genomics, Pleasanton CA, USA). Resulting libraries were pooled and sequenced on the NovaSeq 6000 platform (Illumina, San Diego CA, USA), generating 100-base pair paired-end reads.