Fresh tumor tissues were collected and washed with PBS. The tissues were then cut into approximately 1 mm3 pieces and incubated with a dissociation solution containing DNase I (2 mg/mL; Sigma-Aldrich) and Liberase TL (2 mg/mL; Sigma-Aldrich) at 37°C for 1 h. The dissociated cells were subsequently incubated with 1 × Red Blood Cell Lysis buffer (RBCL; Roche) at room temperature (RT) for 10 min. The cell pellets were collected via centrifugation for 5 min and resuspended in PBS. The suspension was filtered through 40-μm strainers, and single cells were counted using a hemocytometer. The Nextera XT DNA Sample Prep Kit (Illumina) protocol was followed to prepare the sequencing libraries.