GSM5988329: Human_Explant_Macula_Right; Homo sapiens; RNA-Seq
On the 8th day of AAV incubation, the 1mm edges of each explant, which had the highest level of GFP expression, were collected and underwent single-cell dissociation, FACS, and scRNA-seq. The 1mm edge of each retinal explant was placed in Hibernate solution after dissection, and was then dissociated with an enzymatic and mechanical method using a  MACS Neural Tissue Dissociation Kit for postnatal neurons according to manufacturer's recommendations. The macular,  mid-peripheral and peripheral region from the temporal quadrant retina of  human donor#2 was dissected, and the same anatomical regions from left and  right eye were combined and immediately dissociated using the same method.  The retinal single cells were resuspended in D-PBS with 0.1% BSA and  processed immediately for scRNA-seq or FACS. The GFP-positive cells in the  retinal explant cell suspensions were enriched using a MACS Tyto sorter. GFP-positive cells were resuspended in D-PBS with 0.1% BSA and processed immediately for scRNA-seq. Following the manufacture's protocol, using the Chromium Single Cell 3' v3 kit (10x Genomics), single retinal cells were partitioned into Gel Beads in Emulsion (GEMs), mRNA was reverse transcribed to cDNA and barcoded with unique molecular identifiers (UMIs). The GEM emulsions were then broken and the cDNA was purified using DynaBeads. After PCR amplification, the cDNA was cleaned and further purified with SPRIselect reagent The final indexed library was constructed following fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR steps. The libraries were sequenced on Illumina S1 flow cells at the UPMC Genome Center. GFP transcripts with unique AAV barcodes were further enriched using a custom Targeted Gene Expression kit (10x Genomics) and the resulting library was sequenced with an Illumina MiSeq reagent nano kit v2 (300 cycles).