GSM7653320: p21mTert_18M_Rep2_ssRNAseq; Mus musculus; RNA-Seq
Mouse trachea is injected with 1,5 ml dispase 50 U/ml followed by 0,5 ml agarose 1%. Lung is resected and minced in 3 ml DPBS 1x CaCl2+ and MgCl2. Collagenase/dispase 100 mg/ml is added to the chopped tissue and placed on a rotator at 37 °C for 30 min. Enzymatic activity is inhibited by adding 5 ml of DPBS containing 10% FBS and 1mM EDTA. Cell suspension is filtered through 100 μm nylon cell strainer, treated by DNase I and filtered again through a 40 μm nylon cell strainer. Red blood cells are removed by red blood cell lysis treatment for 90s at room temperature. Finally, lung cells are centrifuged at 150g, 4 °C for 6 min, resuspended in DPBS 1x containing 0,02% BSA and counted. Single-cell 3'-RNA-Seq samples were prepared using single cell V2 reagent kit and loaded in the Chromium controller according to standard manufacturer protocol (10x Genomics)