GSM8155297: Adult3, periphery, NeuN; Callithrix jacchus; RNA-Seq
Retinas were dissected in Ames solution (Sigma-Aldrich; equilibrated with 95% O2/5% CO2 for all use) immediately following enucleation. They were dissociated in papain, followed by antibody staining and beads-enrichment or FACS sorting . Foveal and peripheral retina tissues were dissected from neonatal and adult marmoset retinas. The foveal piece (~1.5mm in diameter) of the retina containing the whole fovea area was dissected out and dissociated into individual cells for high throughput droplet-based single cell RNA sequencing (scRNA-seq, 10x Genomics). The peripheral area (any area 5mm away from the foveal center) was also dissociated into a single cell suspension and underwent two enrichment procedures. We first used CD73, a photoreceptor marker, to label photoreceptors and used a microbeads-conjugated secondary antibody to deplete all the CD73 labeled cells (1). Second, we enriched RGCs and ACs with CD90 positive selection via fluorescence-activated cell sorting (FACS). The peripheral cells after CD73 depletion or CD90 enrichment were used to generate scRNA-seq libraries (10X Genomics). Libraries were sequenced on the Illumina HiSeq 2500 platform. The peripheral retinal tissues of an adult marmoset were snap frozen with dry ice. Nuclei were isolated with ice-cold Nuclei EZ lysis buffer and resuspended with 1%BSA/1X PBS. Nuclei were stained with anti-NeuN-PE (Millipore) for 30min at 4C. After washing and resuspension, the nuclei solution was additionally stained with Dyecycle Ruby (Thermo Fisher) before sorting. NeuN-positive and Dyecycle-positive nuclei were sorted via FACS. The sorted nuclei were used to generate single nuclei sequencing (snRNA-seq) libraries (10X Genomics) and were sequenced on the Illumina NovaSeq S4 platform.Frozen foveal and peripheral retinas were retrieved from a -80 °C freezer and placed on ice. Nuclei from these frozen samples were separately isolated using Lysis Buffer and Nuclei Isolation Columns from the Chromium Nuclei Isolation Kit (10X Genomics) following the Manufacturer's User Guide. The isolated nuclei were then resuspended in Resuspension Buffer (10X Genomics). ScATAC-seq libraries were generated from the recovered nuclei using the Chromium Next GEM Single Cell ATAC Reagent v2 Kit (10X Genomics). The quantity and quality of the scATAC-seq libraries were evaluated using Qubit fluorometers (Thermo Fisher) and Tapestation (Agilent). ScATAC-seq libraries were sequenced on the Illumina Novaseq X Plus 10B platform.