GSM8655150: CAR-T; Mus musculus; RNA-Seq
Tumor tissues were excised and mechanically dissociated in ice-cold HBSS supplemented with glucose (10 mM) and HEPES (10 mM) and using glass shearing with a 10-ml Potter-Elvehjem pestle and glass-tube homogenizer (Merck). The resulting suspension was passed through a 70-µm cell strainer (BD Biosciences). The cells were incubated with FC blocking (BD Biosciences). Simultaneously, 20 µl of Cell Multiplexing Oligo (CMO) lipid solution (3' CellPlex Kit, 10X Genomics) was added for multiplexing for 20 minutes. Afterwards, cells were washed with flow cytometry buffer and incubated for additional 20 minutes with anti-CD45 or anti-CD45.2 antibodies (Biolegend). Cell sorting was performed on a MoFlo Astrios (Beckman Coulter). A total of 50,000 cells were sorted per sample and analyzed using 10X Genomics 3' single-cell mRNA sequencing (v3.1 chemistry), resulting in a final yield of approximately 10,000 sequenced cells per reaction. Library preparation were performed according to the user guide for the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (CG000204 or CG000390) with 3' Feature Barcoding kit (10X Genomics, USA) for library preparation of multiplexed samples.