GSM9151613: NHP2, AAV8-GRK1-hRPGR injected, right eye; Macaca mulatta; OTHER
After rapid harvesting of the globes, the cornea and the lens were first carefully removed. The posterior eyecup was flattened with four diagonal radial cuts and the remaining vitreous carefully removed. The flattened eyecup was transferred to a glass slide. Guided by vascular landmarks on retinal images, 4 mm full-thickness retina/choroid/sclera punches were obtained using skin biopsy punches corresponding to treated and untreated areas and numerically assigned for downstream processing. For spatial transcriptomic analysis, full-thickness punches were embedded in optimal cutting temperature compound (VWR, Radnor, PA, USA) and snap frozen in isopentane for storage (at -80 ͦC) or sectioning as per 10X Genomics protocol. Following optimization, retinal punch biopsies from NHP1 and NHP2 previously fixed in PFA were sectioned at 10 μm with a cryostat and positioned on a Visium (10X Genomics) spatial gene expression slide. The Visium slide was fixed in pre-chilled methanol at -20°C for 30 min, and stained with Hematoxylin & Eosin (H&E). Following 10X Genomics protocol, retina sections were incubated for permeabilization for 12 min at 37°C and reverse transcription was performed. Next, second strand cDNA was synthetised and total cDNA was denatured. qPCR was performed using 10X Genomics' specific cDNA primers for cDNA quantification before cDNA amplification and clean-up. The next day, cDNA integrity was measured. Spatial gene expression library was finally constructed and sequenced with Illumina NovaSeq X Plus by Novogene.