GSM6067513: CT2A; Mus musculus; RNA-Seq
Mice were transcardially perfused with 50 ml cold DPBS to remove the blood before isolating tissues. The right hemisphere of the brain was minced into small pieces by blade and digested with 2 mg/ml collagenase IV (Worthington) and 30 μg/ml DNase I (Rhoche) at 37℃ for 30 min. Tissues were passed through a 70 μm strainer. To remove non-immune cells from tumor samples, cells were resuspended in 4 ml of 30% percoll (GE Healthcare) containing DMEM CM and loaded onto 3 ml of 70% percoll containing DPBS supplemented with 1% BCS. Immune cells and myelin were separated by centrifugation. Cells were treated with ACK lysis buffer for 5 min at RT to remove residual red blood cells. Cells were stained with anti-mouse CD45.2-APC or CD45-APC and propidium iodide. PI-negative and CD45.2/CD45-positive live immune cells were sorted with FACS Aria. library were constructed with Single Cell 3' Reagent Kit v3 (10x genomics) following manufacturer's instruction.