GSM7146630: Mouse Lung tumor_KrasLA1_rep2; Mus musculus; RNA-Seq
Briefly, 39 weeks old mice were anesthetized using Avertin (Sigma, T4802), right heart ventricle was perfused with 1x PBS without calcium and magnesium (ThermoFisher, #100100023). Lungs were removed from the mouse and placed in Liebovitz media (Gibco, #21083-023). Lung tumors were identified and collected under the dissecting microscope. Tumors were minced with forceps and digested in the Liebovitz media with 2 mg/mL collagenase type I (Worthington CLS-1, LS004197), 0.5 mg/mL DNase I (Worthington D LS002007) and 2 mg/mL elastase (Worthington ESL LS002294) for 30 min at 37oC. Digestion was stopped with 20% Fetal bovine serum (FBS, Invitrogen 10082- 139). Cells were filtered using a 70 µm cell strainer (Falcon, 352350) on ice and transferred to a 2mL tube. Red blood cells were lysed using Red blood cells lysis buffer (Miltenyl Biotec 130-094-183), and the lysate centrifuged for 1 minute at 5000rpm. Supernatant was removed and cells in the pellet were washed twice with 1mL of ice-cold 1X Leibovitz plus 10% FBS. The cellular suspension was then filtered with a 40 µm cell strainer (Falcon #352340), centrifuged and cells resuspended in ice-cold 1X Leibovitz plus 10% FBS. Cell viability was evaluated using Trypan blue and counted with a hemocytometer. Samples with more than 70% of viable cells were submitted for scRNA-seq After cell dissociation and counting, samples were centrifuged and re-suspended in 1X PBS with 0.04% BSA for single cell suspensions and processed for Chromium Single Cells Gene Expression Solution Platform (10x Chromium) with 3'v3 Library following manufacture instructions.