Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: The NHP retinas were dissected and regions of interest were isolated (macula, superior and inferior periphery). For cynomolgus macaque, superior and inferior periphery were pooled. Retinal tissue was placed in Hibernate solution (Hibernate A -Ca Solution, BrainBits LLC), and cells were then dissociated using Macs Miltenyi Biotec Neural Tissue Dissociation Kit for postnatal neurons (130-094-802) according to manufacturer's recommendations. Dissected retina pieces were incubated with agitation at 37 °C and further mechanically dissociated. The dissociated neural retina was filtered using a 70 µm MACS Smart Strainer (Miltenyi Biotec) to ensure single-cell suspension. Cells were resuspended in 0.1% BSA in D-PBS and processed immediately for scRNA-seq. Brain, heart, and liver of mice were freshly dissected, and cells were dissociated using Macs Miltenyi Adult Brain Tissue Dissociation Kit (130-107-677), Multi Tissue Dissociation Kit 2 (130-110-203) and Liver Dissociation Kit (130-105-807) according to manufacturer's recommendations. The cells were resuspended in 0.1% BSA in D-PBS and processed immediately for scRNA-seq. Following dissociation using Macs Miltenyi Tissue Dissociation Kits specific for retina, brain, heart and liver, a Miltenyi MACS Tyto sorter was used to enrich for GFP-positive cells. Cells were resuspended 0.1% BSA in D-PBS and processed immediately for scRNA-seq. Marmoset and cynomolgus macaque samples were prepared for single cell analysis using a 10x Chromium Single Cell 3' v3 kit. Briefly, single cells from retina samples were captured using 10X Chromium system (10X Genomics), the cells were partitioned into Gel beads-in-emulsion (GEMS), mRNAs were reverse transcribed and cDNAs with 10X Genomics Barcodes were created with unique molecular identifiers (UMIs) for different transcripts. Purified cDNA was PCR amplified and further purified with SPRIselect reagent (Beckman Coulter, B23318). Final libraries were generated after fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR steps according to 10x Single Cell 3' workflow. An additional targeted sequencing analysis was run on these 10x-prepped cDNA samples, using PCR amplification with Q5 High Fidelity DNA Polymerase to target the GFP sequence and its associated AAV barcode. Samples from mouse tissues and cultured 293AAV (Cell Biolabs) cells were prepared for single cell analysis using a 10X Chromium Single Cell 3' v3.1 kit. The resulting libraries were pooled, and an additional targeted gene enrichment protocol was performed using 10X Chromium Targeted Gene Expression kit.