title: GSM6917452: GF, GL261, immune cells, DPBS; Mus musculus; RNA-Seq
library_construction_protocol: To isolate single cells from the mouse GBM TME immediately after euthanasia via a CO2 gas chamber, we perfused 30 ml of cold DPBS solution through the heart to prevent blood contamination in the brain as much as possible. Then, only the right hemisphere, in which the cancer cells were inoculated, was extracted. The hemisphere was divided into small pieces using a blade and digested with 2 mg/ml collagenase IV (Worthington Biochemical Corporation, Columbus, OH, USA) and 30 μg/ml DNase I (Roche, Basel, Switzerland) at 37 °C for 30 minutes in a shaking incubator. Next, the tissues were filtered through a 70-µm strainer. To isolate immune cells, we resuspended the filtered cells in 5 ml of 30% Percoll solution (GE Healthcare, Chicago, IL) containing DMEM CM and then a 70% Percoll solution containing DPBS supplemented with 1% bovine serum (Thermo Fisher Scientific) was added slowly to the bottom, followed by centrifugation at room temperature for 20 minutes without applying a brake. After removing the immune cells at the interface of the two Percoll layers, the cells were incubated in ACK lysis buffer at room temperature for 5 minutes to remove the remaining red blood cells, and further analysis was performed. Suspensions were pooled into one sample from three mice in each group with a median of five mice. The single-cell suspensions were treated with an anti-CD16/32 antibody (2.4G2, Fc blocker) to block Fc receptors prior to staining with mouse-specific antibodies. The cells were then stained with CD45.2-PE. Live immune cells were isolated using a FACS Aria II (BD Biosciences) flow cytometer, and 7-AAD staining was performed before data acquisition to eliminate dead cells and 7-AAD-negative and CD45.2-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.