Single cell RNAseq of 12-week old AKIMBA versus WT mouse retina
Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2AkitaxVEGF+/-) mice, which are known to replicate features of clinical diabetic retinopathy. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by measuring the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina.  Retinas (n=4 for Akimba, n=2 for wild-type) were isolated in ice-cold Dulbecco's Modified Eagle Medium. After rinsing with Dulbecco's Phosphate-Buffered Saline containing 2% fetal bovine serum, each retina was incubated with 1mL digestion buffer (2mg/mL collagenase-P, 200U/mL DNAse-I (Sigma-Aldrich) in M199 medium (Life Technologies) at 37°C for 10min. Retinal tissue was further dissociated by trituration and the suspension was filtered through a 40µm cell strainer and centrifuged for 5min at 300xg (4°C). Pooled retinal single-cell suspensions from wild-type and Akimba were counted on a Luna-FL Cell Counter (Logos Biosystems) and libraries were prepared with the Chromium Single-cell 3' V2 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics) aiming for 5000 cells per library. Barcoded libraries were sequenced on an Illumina HiSeq4000 in 25-8-98 paired-end configuration.