GSM8751544: 3-day ligated sample 2; Mus musculus; RNA-Seq
For enrichment of stromal cell populations, tThree mice were used for each timepoint with the mouse's right submandibular and sublingual gland both being harvested. Excess fat and interstitial tissue were removed. The glands were then transferred to a dish containing 1X PBS, liberase TL Research Grade low Thermolysin (Roche 05401020001) at 0.25 mg/ml, DNase I (Stemcell Technologies 07900) at 15mg/ml, and dispase (Gibco 17105-041) at 0.53 u/ml and microdissected for 10 minutes. The sample was then incubated in a 37°C incubator for 15 minutes then triturated 1200 times . 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 containing equal volume of DMEM/F12 (Gibco 11039-021) with 10% FBS (Gibco 10082-147) and was centrifuged for 5 minutes at 150xg 4C. The supernatant was removed and discarded, and the cell pellet was resuspended in 2 ml of isolation buffer composed of sterile-filtered Ca2+- and Mg2+-free 1X PBS, 0.1% bovine serum albumin, and 2 mM ethylenediaminetetraacetic acid. 5 μg of EpCAM (Invitrogen 14-5791-81) and Ter119 (Invitrogen 14-5921-82) was added to the cell suspension and incubated at 4°C on a rocker set on low speed for 10 minutes. the cell suspension was washed using isolation buffer and centrifuged at 150xg for 5 minutes at 4C. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of isolation buffer. For depletion of epithelial and red blood cells, 25 μL of sheep anti-rat Dynabeads, which bind to the EpCAM- and Ter119 Ter119-labeled cells, (Invitrogen 11035) were added. The cell suspension was transferred to a 35 mm dish before incubating at 4C for 20 minutes on a rocker set at low speed. The sample was placed on a microcentrifuge magnet for 2 minutes, and the supernatant was collected. One additional epithelial and red blood cell depletion was performed. The sample was then depleted of dead cells using a dead cell removal kit (Miltenyi Biotec 130-090-101), following manufacturer's instructions. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics).