Single-cell RNA sequencing analysis of mouse normal brain and GBM tumors

To investigate the transcriotome in glioblastoma tumor and normal brain on a single cell level, we performed RNA sequcening analysis with single cells derived from mouse normal brain tissue or GBM tumor tissue. Particularly, vascular endothelial cells (ECs) were traced using a Cdh5-Cre;LSL-tdTomato system. Chicken DF-1 fibroblasts were transfected with RCAS-PDGF-B and RCAS-Cre plasmids to produce retrovirus, followed by orthotopic injection into Ntv-a;Ink4a-Arf-/-;Ptenfl/fl;LSL-luc mice to induce GBM. Tumors were isolated and subjected to mechanical dissociation with a gentleMACS Dissociator and enzymatic digestion using collagenase II and dispase II to obtain single-cell suspensions. Tumor-derived single-cell suspensions were cultured with NeuroCult mouse stem cell medium. Attached cells were discarded to remove tumor stromal cells, and non-adherent sphere-forming tumor cells were injected into Cdh5-Cre;LSL-tdTomato mouse brains.