GSM8208508: Ace2 KO mouse corneal rim 2; Mus; RNA-Seq
Whole eyes were harvested . The lens, retina, iris, and trabecular network were removed leaving the entire ocular anterior segment, from which most of the conjunctiva was carefully dissected and discarded. The corneal rims were subsequently digested at 37°C for 2 hours with 2 ml of 1 mg/mL collagenase A (Sigma-Aldrich) in DMEM containing 10% FBS, 50 μg/mL gentamicin, and 1.25 μg/mL amphotericin B.  After 2hr incubation, the corneal rims were rinsed once with PBS without Ca2+-Mg2+. Clusters were further digested with 0.25% trypsin and 1 mM EDTA at 37°C for 20 minutes with intermediate gentle pipetting to yield single cells. After trypsin digestion, cells were rinsed once with DMEM+10% FCS media to deactivate and remove trypsin. The cells were rinsed twice with sterile PBS containing 0.04% BSA. After final centrifugation, cells were resuspended in 60ul of PBS containing 0.04% BSA to achieve 700-1200 cells/ul, and filtered into an Eppendorf tube by using Flowmi Cell Strainer (Fisher Scientific). Cell viability was checked and accepted with at least an 80% viability score. Such cells were processed for scRNA-seq using 10XGenomics pipeline in the Northwestern University NUseq Core. follow the instructions from 10XGenomics