title: GSM8262101: Old, SV, scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: After euthanizing the mice, the urogenital system was exposed. The entire urogenital system was taken out of the mouse abdominal cavity and placed into a 60 mm Petri dish containing 5 ml complete medium (CM) composed of IMDM (Invitrogen), 10% FBS (Sigma), and Antibotica-Antimycotic (invitrogen). Under a dissection microscope, carefully remove the surrounding organs and tissues. Open the seminal vesicle longitudinally with a scissor and let seminal vesicle fluid out of the organ. Transfer the tissue to another dish and gently remove any loose coagulated seminal vesicle fluid without disturbing the epithelial cell layer.  After washing, the tissue was transferred into a 15 ml conical tube with 2 ml CM and 2 ml extracellular matrix digestion media (ECM) containing collagenase (Sigma), DNase (Sigma), collagenase/hyaluronidase (Stem Cell), and collagenase/dispase (Sigma). The tissue was incubated in a water bath at 300 rpm for 30 minutes at 35 C. After incubation, 10 ml IMDM-DNase media was added to the tube and centrifuged at 300 g for 5 minutes at room temperature. Wash the pellet once with 8 ml IMDM-DNase media. Resuspend the pellet with 3 ml TrypLEtm express (Gibco), and digested the tissue in the water bath at 300 rpm for 5 minutes at 35 C. Fill the tube with 10 ml CM and filter the cell suspension through a series of 100 μm, 70 μm, and 40 μm cell strainers. Pellet the cells by centrifuging for 5 minutes at 300 g. Resuspend the pellet with 3 ml CM. The cell suspension was then placed on the top of OptitPrep (Sigma) gradient followed by centrifuging at 800 g for 15 minutes. The gradient was composed of 3 ml of 67%, 50%, and 36% OptiPrep in CM, respectively. Interface fractions were collected and cells were pelleted, washed 1X with CM, and resuspended in 1 ml CM. The samples with cell viability over 80% were used for sequencing library preparation. Single-cell sequencing libraries were prepared using Chromium Single Cell 3' Reagent Kit V2 (10X Genomics), as per the manual.