title: RNA-seq of mus musculus: Tox Experiment with 4KR
design: Experiment workflow For the transcriptional profiling of Tex cells, we established a B16-OVA melanoma model to analyze tumor antigen-specific CD8+ T cell exhaustion. Briefly, OT-I CD8+ T cells were activated in vitro using anti-CD3 and anti-CD28 antibodies. On day 9 following the implantation of B16-OVA tumors, 3  106 OT-I CD8+ T cells were adoptively transferred to each tumor-bearing mouse. At day 14 after the adoptive transfer, cells were sorted from tumors and spleens using flow cytometry. PD-1 and TIM-3 were used to label different subsets of exhausted TILs: the PD-1-TIM-3- population, PD-1+TIM-3- population and PD-1+TIM-3+ population. For the transcriptional analysis of adoptively transferred WT and Klhl6-/- CD8+ OT-I T cells in the tumors, Klhl6-/- CD8+ OT-I T cells (CD45.1/2+) and WT CD8+ OT-I T cells (CD45.1+) were mixed in a 1:1 ratio and adoptively transferred into the same B16-OVA tumor-bearing mice. After 14 days, the transferred CD45.1+ and CD45.1/2+ CD8+ T cells were sorted from tumors using flow cytometry and prepared for RNA extraction. Total RNA from the isolated transferred OT-I T cells was extracted using the RNeasy Micro Kit (Qiagen) following the manufacturers instruction and stored at -80 C for RNA sequencing. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent). Subsequently, the libraries were prepared using the TruSeq RNA sample prep kit (FC-122-1001, Illumina). These libraries were then subjected to sequencing on an Illumina NovaSeq 6000 (PE150) platform, generating approximately 40 million paired-end reads (Novogene).