Illumina HiSeq 4000 sequencing; Single cell RNAseq of 12-week old AKIMBA versus WT mouse retina
Single cell RNAseq of 12-week old AKIMBA versus WT 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)  Single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3' or 5' Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 4,000 cells per library.  Samples were processed using 10X Genomics V2 chemistry kits
Experimental Factor: genotype
wild type genotype