GSE125698
Gene expression in B16.SIY tumor cells deficient in IFN-g signaling
We performed RNASeq on purified tumor cells from wild-type, IFNgR2-, and Jak1-mutant tumors on day 7 after tumor engraftment. To investigate whether IFN-g-insensitive tumor cells showed decreased expression of a negative immune regulatory factor, RNASeq was performed on purified tumor cells from WT, IFNgR2-, and Jak1-mutant tumors on day 7 after tumor engraftment.  Compared to wild-type, we found 375 and 305 differentially expressed genes in IFNgR2- and Jak1-mutant tumor cells respectively. 228 of these genes were shared between IFNgR2- and Jak1-mutant tumor cells. Overlapping downregulated genes included those involved in antigen presentation, immune functions, extracellular matrix, ubiquitination, and GTPase activity.RNA sequencing and alignment to gene features were performed by the University of Chicago Genomics Core facility using the Illumina HiSeq platform. Raw read counts were processed by TMM normalization followed by log2 transformation. Genes expressed in fewer than three samples were removed from further analysis. Limma voom was used to identify differentially expressed genes between each mutant cohort and the wild-type cohort with a cutoff fold change > 2 and adjusted p-value < 0.05. Overall design: Wild-type, IFNgR2-, and Jak1-mutant tumor cells were inoculated into C57BL/6 mice and tumors were extracted 7 days later. Tumors were enzymatically digested for 30 min at 37oC with rotation. Tumor suspension was Ficolled and buffer layer was washed twice with PBS. Cells were stained with CD45, CD3, Thy1, CD19 antibodies and a live-dead permeability dye to gate out hematopoietic cells and dead cells. Tumor cells were cell sorted based on dsRed and BFP expression into RLT buffer and quickly frozen.  RNA was extracted using the RNEasy micro kit (QIAGEN) and submitted for single-end RNA sequencing.