title: GSM8893251: P5 Retina Cx3cr1-GFP+ nuclei; Mus musculus; RNA-Seq
library_name: GSM8893251
library_construction_protocol: For scRNA-seq, eyes from Cx3cr-CreER-ires-YFP mice were enucleated into ice cold oxygenated AMES media. Tissues and cells were kept on ice throughout the procedure except as noted, and centrifugation was carried out at 4˚C. To isolate the retina, the anterior portion of the eye was removed and the retina was dissected free of the eyecup. The retinal pigment epithelium typically remained partially attached upon removal from the eyecup. All YFP-positive mice in each litter were used, and retinas were pooled for subsequent steps. Retinal tissue was dissociated in a solution of activated papain (Worthington, 16 U/mL) at 37˚C for 25 to 30 min, then triturated in an ovomucoid solution (Worthington, 15 mg/mL) to prepare a single cell suspension. The suspension was passed through a 30 μm cell strainer and pelleted at 400 g for 7 min. The cells were resuspended in AMES solution and incubated in LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo, 1:1000) for 30 min at 4˚C. Cells were washed twice, centrifuged at 400 g for 2 min, and resuspended in 0.4% BSA in 1X PBS. The YFP+ live population was collected via flow cytometry (MoFlo Astrios or BD Diva cytometers) and sorted into 0.4% BSA in 1X PBS. Post-sorting, samples were pelleted and resuspended in PBS + 0.04% BSA. For snRNA-seq, animals of genotype Cx3cr1CreER; Rosa26-loxStoplox-Sun1GFP were injected with tamoxifen at P0-1 and collected at P5 or P10. An entire litter of mice labeled in this manner were used at each timepoint. Retinas were isolated in ice-cold oxygenated AMES media, flash-frozen in liquid nitrogen, and stored at -80 degrees C until processing. On the day of sorting, nuclei were isolated as follows: Retinas were thawed for 5 min on ice; incubated for 5 min on ice in lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl; 3 mM MgCl2; 10% Nonidet-P40 substitute; 40U/µL RNAse inhibitor); triturated 20 times with a wide-bore pipette tip; incubated on ice an additional 10 min; and homogenized with 20 strokes of a disposable microcentrifuge tube dounce homogenizer. The preparation was then suspended in 0.5 mL AMES + 2% BSA containing 40U/µL RNAse inhibitor; passed through at 30 µm cell strainer; and pelleted at 500 g for 5 min at 4˚C before resuspending in AMES + 2% BSA. GFP+ nuclei were isolated by flow cytometry and resuspended in PBS + 0.04% BSA. Samples were processed for 10x Genomics droplet encapsulation and library construction by the Duke Molecular Genomics Core. The 10x Genomics 3' single cell assay v3 kits were used, according to the manufacturer's instructions.