GSM7507533: homeostatic stroma-enriched murine  submandibular salivary gland_1; Mus musculus; RNA-Seq
Two deligated or homeostatic submandibular and sublingual glands were harvested, and excess fat and interstitial tissue was removed. The glands were then transferred to a dish containing 1X PBS, liberase TL Research Grade low Thermolysin (Roche), DNase I, and dispase and microdissected for 7-9 minutes. The sample was then incubated in a 37°C incubator for 15 minutes and triturated. After trituration, the sample was incubated for an additional 5 minutes in a 37°C incubator and triturated once more. A 15 mL conical tube was placed on ice and the entire sample was transferred to the conical tube and incubated for 10 minutes. The supernatant was isolated and transferred to a fresh conical tube and was centrifuged for 5? minutes at 450xg. The supernatant was removed and discarded, and the cell pellet was resuspended in isolation buffer composed of sterile-filtered Ca2+- and Mg2+-free 1X PBS, 0.1% bovine serum albumin, and 2mM ethylenediaminetetraacetic acid. 5 mg of EpCAM-A647 (catalog: 118212, BioLegend) and Ter119 (catalog: 50-133-27, eBioscience/ThermoFisher) was added to the cell suspension and incubated at 4°C for 10 minutes for depletion of epithelial and red blood cells, respectively. The cell suspension was washed using isolation buffer and centrifuged at 450xg for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of isolation buffer and 25 uL of sheep anti-rat Dynabeads, which bind to the EpCAM and Ter119 labeled cells, (catalog: 11035, Invitrogen) before incubating on ice for 20 minutes on a rocker. The sample was placed on a microcentrifuge magnet for 2 minutes, and the supernatant was transferred to a fresh microcentrifuge tube to remove labeled epithelial and red blood cells. An additional epithelial and red blood cell depletion was performed. The sample was then depleted of dead cells using a dead cell removal kit (catalog: 130-090-101, Miltenyi Biotec) following manufacturer's instructions. Cells were counted and resuspended to a concentration of 1000 cells/mL. Homeostatic libraries were prepared in two technical replicates, while the deligated libraries were from two biological replicates. Following the manufacturer's protocol for the Chromium Next GEM Single Cell 3' Reagent kits v3.1, scRNAseq libraries were generated. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics).