GSM8463762: Corneolimbal, 18 week old, Confetti- 55; Mus musculus; RNA-Seq
Male and female Confetti mice, aged 5, 17 and 59-weeks (n=3/age), were administered Tamoxifen (5 mg/mL) (Sigma-Aldrich, St Louis, MI) in 10% ethanol (Sigma-Aldrich) and 90% sunflower seed oil (Sigma-Aldrich) by intraperitoneal injection on three consecutive days to induce expression of the multi-color fluorescent reporter. Seven days after the first injection, mice were euthanized by cervical dislocation, and their eyes immediately enucleated. Corneas, complete with the limbal ring, were dissected under a stereo microscope (Zeiss, Oberkochen, Germany), taking care to remove extraneous tissue. Samples were exposed to 0.5 U/mL Dispase II (Penzberg, Germany) in defined keratinocyte serum-free media (dKSFM) (ThermoFisher Scientific, Waltham, MA) for 2 hrs at 37°C, then trypsin-EDTA (ThermoFisher) for 10 min at 37°C to generate a single-cell suspension of corneolimbal epithelia. Cells underwent FACS for colored Confetti+ (CFP, GFP, YFP, RFP) and non-colored Confetti- populations, taking care to only sort the epithelial population based on cell granularity, size and shape, and minimizing other cell types such as immune cells. Single-cell libraries were prepared using the SmartSeq-2 protocol, and sequencing was performed on the Illumina NextSeq platform (Ramaciotti Centre for Genomics, UNSW, Sydney, Australia). Mouse corneolimbal epithelia (n=378 total), including Confetti+ and Confetti- cells (n=189/group) were segregated according to three time-points corresponding to mouse age (n=63 cells/time-point) (T1=6, T2=18 and T3=60-weeks), then sequenced and analyzed. Reads were quantified with Salmon (v0.9.1) using the mouse transcriptome GRCm38 to create an index before quantification. Read counts ranged from 288,000-1.4 million/cell with a median of 778,000/cell. The number of genes per cell ranged from 546 - 7,246 with a median of 4,758. Reads were summarized according to gene level using the R package Tximport [103]. The R package Seurat (v3.1.4) [104] was used to filter and normalize data, after which clustering and differential expression analysis were conducted. Cells with >5% mitochondrial content and those with <2000 genes were then removed, and the number of cells analyzed for Confetti+ at T1, T2, and T3, and Confetti- at T1, T2, and T3 were 62, 55, and 41, and 61, 60 and 59, respectively. After filtering and normalization, 14,343 genes were identified across the data set in 338 cells.