GSM5241830: Mouse embryonic retinal progenitor cells - E14.5 DHS4D-GFP+mCherry; Mus musculus; RNA-Seq
Embryonic mouse retinas were cultured up to 48hrs following electroporation and then dissociated using the Worthington Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, New Jersey, USA). Tissue was placed into activated papain tubes, prepared via manufacturer specifications, and incubated on a nutator at 37°C. Dissociation status of tissue was checked every 10 minutes and the cells gently triturated using a 1mL pipette 2-3 times. Just prior to achieving a final single cell suspension, warmed DNase solution was added dropwise, gently inverted to mix, and then incubated for 5 more minutes on rotator at 37°C. Next, prewarmed ovomucoid solution was slowly added dropwise and again gently inverted to mix. Cells were spun down for 5 minutes at 300 g and the supernatant carefully aspirated. Cells were then resuspended and washed with 1% FBS + PBS (without calcium or magnesium). After washing, cells were spun 5 minutes at 300 g and resuspended with 1% FBS + PBS for fluorescently activated cell sorting. GFP+(±Cherry) and Cherry-only cells were separately collected from cell sorter into 10% FBS+PBS (without calcium or magnesium), counted on a hemocytometer, and resuspended in 10% FBS + PBS. To generate sequencing library GFP+ (±Cherry) and Cherry-only cells were mixed at a 40/60 ratio. Single cell suspension was then submitted for droplet based barcoding on the 10X Chromium using the Chromium Single Cell 3' Reagent Kit V2 (10X Genomics, Pleasanton, California, USA). 2x151 scRNAseq