GSM8409444: WT 7wk; Mus musculus; RNA-Seq
To ensure biological diversity, nuclei were initially isolated from snap frozen corneas of 5 mice per treatment group at 7 wks of age (WT,KO,PBS). Nuclei isolation protocol was adapted from 10x Genomics (Sample Preparation Demonstrated protocol, Rev D). Briefly, fresh corneas were dissected, minced, snap frozen in lysis buffer (Sucrose 0.32M, CaCl2 3mM, Mg(Ac)2 3mM, EDTA 0.1mM, Tris-HCL 10mM, DTT 1mM, Triton x100 0.1%, DEPC-treated water) and stored for further processing. Upon nuclei isolation, frozen sample were crushed using a pastel and combined into 15ml tube, with total volume of 5ml lysis buffer. Samples kept on ice for 15min with “vortexing” every 5 min. Sample were then transferred into ultracentrifuge tubes on ice (Beckman Coulter 355631), and 9ml of Sucrose solution (Sucrose 1.8M, Mg(Ac)2 3mM, Tris-HCL 10mM, DTT 1mM, DEPC-treated water) were carefully added to the bottom of the tube. Sample were spined 24,400 rpm for 2.5hr at 4°c. After spin, supernatant was removed, and 200ml of DEPC-based PBS was added for 20 min incubation on ice followed by resuspension. isolated nuclei solution was filtered twice using 10mm filter. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics).