GSM9144819: Tumor, EGFRvIII CAR T-cells, Biological replicate 2; Mus musculus; RNA-Seq
C57BL/6 mice were orthotopically injected with 6x105 hCD19+ murine PDAC cells (KPC cell line), followed by intraperitoneal injection with murine CAR-T cells targeting either hCD19 or a control epitope (human EGFRvIII). CAR T-cell treated tumors were isolated after 10 days for control CAR T-cell treated mice and after 15 days for hCD19 CAR T-cell treated mice, at which stage all PDAC tumors had reached a similar weight. The tumors were isolated and dissociated into single cell suspensions. For this purpose, tumors were minced with a scalpel and incubated for 30 min in a HBSS-based digestion buffer containing 10 mM HEPES (Invitrogen, #15630080), 12.5U Collagenase IV (Millipore Sigma, C2674), and 200U DNaseI (NEB, #M0303) at 37°C. Next, tumors were further dissociated using a gentleMACS tissue dissociator (Miltenyi Biotec). Enzyme-activity was quenched with FBS and the samples were filtered through sequential filtering steps to obtain single cell solutions. 10,000 live cells were submitted in PBS with 5% FBS for transcriptional profiling. Single-cell expression libraries were prepared using the 10x Genomics Chromium v3 reagents. Library construction was performed according to the 10X Genomics Chromium v3.