Summary: Here, we present a protocol for exploring the effects of PPP1R15A inhibitor, Sephin1, on antitumor immunity of B16F1 subcutaneous tumor in mice. We describe steps for constructing single-cell transcriptome and TCR libraries, sequencing, and using sequencing data for the integration of expression and TCR data. We then detail procedures for gene differentiation, regulon and cell-cell communication analysis, and validation of single-cell analysis results.For complete details on the use and execution of this protocol, please refer to Wang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.