GSM8144089: Paraspinal tumor, PD-1540; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane, perfused with ice cold phosphate-buffered saline (PBS) lacking calcium and magnesium. Paraspinal tumors were collected within 5 minutes of death and stored in L-15 medium until tissue was cut into 1-mm3 pieces and placed into dissociation medium in 50 mL tubes containing 20 mL L-15 medium, 10 mg collagenase type I, and 50 mg Dispase II in a 37°C incubator for 2 hours, with 170 rpm shaking. The dissociation process was stopped by adding 30 mL DMEM containing 10% FBS. Samples were centrifuged at 800g for 5 minutes at 4°C. Supernatants were removed and cell pellets were resuspended into 50 mL of 1× PBS containing 0.1% BSA. Suspensions were sequentially filtered with 70 μm, 40 μm, and 20 μm cell strainers and centrifuged at 600g for 5 minutes at 4°C. Pellets were resuspended into 100 μL of 1× PBS/0.1% BSA. Trypan blue–negative viable cells were counted to determine the cell concentration for droplet-based scRNA-seq. Library was performed according to the manufacturer's instructions (single cell 3v3.1 protocol, 10x Genomics)