GSM9843634: Control cornea, WT-1; Mus musculus; RNA-Seq
The eyes from 10 wild type (WT) mice and 10 Dot1l-/- mice (Dot1l-KO) were enucleated, and the corneas were carefully dissected and rinsed 2–3 times with ice-cold sterile PBS. Corneas were incubated with Dispase II (Sigma-Aldrich, 15 mg/ml) at 4 °C for 16 h to separate the epithelial layer. Following enzymatic treatment, the corneal epithelium was gently peeled away from the underlying stroma using ribbed forceps and subsequently dissociated with 0.25% trypsin/EDTA for 10 min. The remaining stromal tissue was digested with collagenase A (Sigma-Aldrich, 2.5 mg/ml) at 37 °C for 1 h. Digestion was terminated by adding an equal volume of DMEM supplemented with 10% fetal bovine serum (FBS). The resulting cell suspensions were filtered through a 40 µm cell strainer to remove large aggregates and debris. Cell viability and quality were assessed using a Luna automated cell counter, with acceptance criteria set at viability > 80%, clumping rate < 5%, and debris/fragment content < 5%. Following quality control, single-cell suspensions from WT and Dot1l-/- groups were individually pooled, resuspended, and adjusted to a concentration of 700–1200 cells/µL in preparation for 10 × Genomics single-cell RNA sequencing. In brief, single cell was loaded onto a Chromium Single Cell Chip (Chromium Single Cell G chip Kit, 10 x Genomics, PN-1000120) to generate single-cell GEMs and construct barcoded cDNA libraries using the Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10 x Genomics, PN-1000268) following the manufacturer's instructions. The quality of the resulting libraries was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies), and sequencing was performed on an Illumina platform (Annoroad Gene Technology Co., Ltd., Beijing, China).