GSM9257759: 7250 OS retina; Sus scrofa; RNA-Seq
Two eyes per condition (vehicle and AAV2.7m8/CAG-GFP at weeks 2 and 4) were individually processed for single-nuclei RNA sequencing. After collecting aqueous and vitreous humor samples, whole globes were flash-frozen in liquid nitrogen, followed by retinal dissection and freezing. Nuclei were isolated using the 10x Genomics Nuclei Isolation Kit with modifications, including tissue homogenization in ice-cold lysis buffer and 10-minute incubation on ice. The nuclear suspension was stained with DAPI (5µM) during the first wash step and incubated for 20 minutes at 4°C with rotation. Finally, nuclei were purified using fluorescence-activated nuclear sorting based on DAPI fluorescence and collected in wash and resuspension buffer containing PBS, BSA, and RNase inhibitor. Sorted nuclei event count and final volume was used to inform sample loading volume, and the nuclei were immediately loaded onto 10x Chromium GEM-X Single Cell 3' Chip Kit v4, at ~20k nuclei per sample (this loading concentration was optimized based on sorter nuclear count and 10x nuclear recovery). Four lanes per sample were loaded to maximize cell recovery. Libraries were generated following the manufacturer's protocol and were sequenced using the Illumina NextSeq 2000.