GSM4008318: ANTSi_scRNAseq; Mus musculus; RNA-Seq
After euthanasia of mice, eyes were removed and retina dissected out and isolated from vitreous and retinal pigmented epithelium. Retinas were then dissociated in a Papain and DNase I solution for 20 minutes at 37 ºC on a nutator. After incubation, retinas were gently triturated to generate a single-cell suspension, followed by the addition of Ovomucoid. Cells were then spun down at 300 g at 4 ºC then resuspended in Neurobasal medium, passed through a 35 mm filter, and transferred to a BSA coated tube prior to FACS-purification. FACS was performed on a BD FACSAria III Cell Sorter (BD Biosciences) and the GFP-positive fraction was collected for single-cell RNA-seq. 44,000 cells in total were captured for single-cell RNA-seq. Four ANTSi-treated mice (8 eyes) had their retinas pooled for FACS-purified cells and were spun down at 300 g at 4 ºC then resuspended at a concentration of 1000 cells per L in a 0.04 % BSA in PBS solution. Cells were processed through the 10x Genomics Single Cell 3' Chip and processed through the standard Chromium Single Cell 3' Reagent Kits User Guide protocol with a target capture of 4,000 cells. Library QC was determined by Bioanalyzer (Agilent). Libraries were then sequenced on Illumina NextSeq 500/550 vs kit and reads were processed through 10x Genomics Cell Ranger pipeline.