Single-cell RNA sequencing (scRNA-seq) of TIICs from Hepa1-6 HCC tumors

For the CON, LDRT, DPVB, LR-DPVB groups, 3 independent single-cell suspensions were evaluated for each group (12 samples in total). Individual single-cell suspensions of 12 samples were separated by flow cytometry sorting (BD FACS Aria II) from single-cell suspensions mixed with 12 samples according to GFP or tCD19 tags, followed by scRNA-seq processing. According to the protocol, cell suspension was loaded onto RhapsodyTM Cartridge (BD) using BD RhapsodyTM Cartridge Kit (BD, Cat# 633731) and BD RhapsodyTM Cartridge Kit (BD, Cat# 633733) to generate single-cell magnetic beads in microwells, that is, individual cells were suspended in sample buffer (BD). The captured cells were lysed, then the released RNA was barcoded by reverse transcription in individual microwells. cDNA was generated after 45 min of reverse transcription on a ThermoMixer®C (Eppendorf) at 1200 rpm and 37 °C, followed by amplification and quality assessment using an Agilent 4200. scRNA-seq library was constructed using BD RhapsodyTM WTA Amplification Kit (BD, Cat# 633801) according to the manufacturer’s instructions, and finally sequenced using Illumina Novaseq6000 sequencer.