scRNA-Seq data generation

The tumors and adjacent non-cancer tissues were cut into small pieces (approximately 1~2 mm³) in the RPMI-1640 medium (Gibco, Cat #26140079) with 10% fetal bovine serum (FBS, Gibco, Cat #21875034), and digested by Tumor Dissociation Kit (Miltenyi Biotec, Cat #130-095-929) using gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec, Cat #130-096-427), according to the manufacturer’s instructions. The dissociated cells were subsequently filtered by 100 µm SmartStrainer and treated with red blood cell lysis buffer (Miltenyi Biotec, Cat #130-094-183) on ice for 1~2 minutes to lyse red blood cells. After being washed twice with 1×PBS (Gibco) and centrifuged at 400×g for 5 minutes each time, the cell pellets were resuspended in the FACS (Fluorescence Activating Cell Sorter) buffer (PBS supplemented with 2% FBS) at the approximate concentration of 10⁶ cells/mL, and then stained with the antibody against human CD45 (ThermoFisher, Cat #11-0459-42, RRID: AB_10852703) and 7-AAD (ThermoFisher, Cat #00-6993-50). CD45⁺ living cells were sorted by flow cytometry with a BD FACSAria™ III sorter (BD Biosciences, USA) for sequencing. 10,000~20,000 cells were used for the 10x Chromium Single-cell 5’ and VDJ library construction (10x Genomics, USA), according to the manufacturer’s instructions. Purified libraries were subject to Hiseq X Ten sequencer (Illumina, USA) for 150-bp paired-end sequencing.