Single-cell gene expression data from immunocompetent glioma-bearing mouse brain with CD8tm.41BB.z CAR
All animals used for tumor implantation were 10- to 12-week-old males or females. For intracranial implantation, mice were anesthetized and placed in a stereotactic rodent surgery platform. A 1-mm burr hole was drilled into the skull at 1 mm anterior and 2 mm to the right of the bregma. A total of 1x105 GL261 cells in 2 L of media were injected at 3-mm deep as described in 4. Wound clips were then used to close the surgical site. On Day 7 post tumor implantation, animals were treated with 3x106 effector cells adjusted to 40% CAR expression and suspended in 2 L of media via intra-cranial injection using the same burr hole coordinates. Brain tumor samples were harvested after mice were humanely killed via CO2 inhalation followed by decapitation. Briefly, brains were excised from the skull and the upper right quadrants of each brain including the tumor and surrounding normal tissue were collected in 2 mL of RPMI with 5% FBS. Brain tumor samples were then minced with scissors and incubated with 1mg/ml of collagenase IV (STEMCELL, Cambridge, MA) and 50 U/ml of DNase I (Fisher Scientific, Hampton, NH) for 1 hour at 37C. After enzymatic digestion, samples were then pushed through a 70 m cell strainer with a syringe plunger to create a single cell suspension. The samples were then centrifuged, and the pellet was suspended in 10 ml of RPMI with 5% FBS. Cells were first stained with Fc Block (Biolegend, San Diego, CA) for 10 minutes at 4 C. Antibodies  were then added, and cells were incubated for 30-minutes at 4C followed by washing with staining buffer. DAPI was used for dead cell staining prior to sorting and two populations were sorted (CD45- TER119- and CD45+ TER119-) into RPMI media with 10% FBS. Viable CD45+ and CD45- populations were mixed at 70:30 ratio and processed on the Chromium system (10X Genomics, 3 v3) with a target of at least 10,00 cells per library reaction.