scRNA-Seq of mus musculus: adult cornea: CyclinB1-GFP positive cells
Dissected corneas from 15 mice (n = 30) were placed into 2.4 units/l of Dispase prepared in Keratinocyte Serum Free Medium and incubated overnight at 4C. For each cornea, the epithelium was gently peeled off, washed in 1 X PBS, and incubated in Trypsin-EDTA (0.25%) at 37C for 20 minutes. Following incubation, tissue was processed into a cell suspension via successive rounds of manual pipetting with a P1000 tip. 2 ml of FBS and 6 ml of 1 X PBS were added to the cell suspension and cells were spun down at 1000-1500 rpm for 5 minutes. Following centrifugation, the supernatant was aspirated, cells were resuspended in 1X PBS, and the entire volume was filtered through a 100 m cell strainer to obtain a single cell suspension. For cell staining, DAPI (1:1000) was added directly to the cell suspension and incubated for 10 minutes on ice. Following incubation, cells were spun down at 1000-1500 rpm for 5 minutes, supernatant was aspirated, and cells were resuspended for sorting in 2% FBS 5 mM EDTA in 1X PBS. Flow cytometry was performed on a BD LSRII cytometer (BD Biosciences), sorting on a BD FACS Aria II sorter (BD Biosciences). Only GFP+ live cells were collected from sorting. Flow cytometry data was collected and exported using BD FACs Diva software (BD Biosciences) and analyzed and plotted using FlowJo software. The FACS-generated cell suspension was used to generate a single-cell RNA-seq library according to the 10x Genomics protocol (Chromium Single Cell 3 Library & Gel Bead Kit v3, PN-1000075). Single cell separation was performed using the Chromium Single Cell B Chip Kit (PN-1000074). The library was sequenced on an Illumina NextSeq500 instrument with a high-output mode (Illumina, 20024907) 150bp paired-end reading strategy according to 10X recommendations.