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Summary: Understanding the heterogenicity of tumor-infiltraing lymphocyte (TIL) populations and the immunobiology in human cancer is a key to establish efficient immunotherapies. Here, we have established a protocol for the characterization of CD8+ TILs in tumors by single-cell RNA-seq paired to VDJ profiling and chromatin structure including dissociation of tumor biopsies. We have also provided guidance for subsequent fluorescence-activated cell sorting (FACS), single-cell encapsulation, bioinformatics analysis, and troubleshooting.For complete details on the use and execution of this protocol, please refer to Anadon et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.