RNA-seq analysis of murine Sox4+/+ versus Sox4-/- 4T1 TNBC cells

We identified the SOX4 transcription factor and integrin alpha V encoded by the ITGAV gene as important resistance mechanisms to T cell-mediated cytotoxicity of TNBC cells. Here, we performed RNA seq. analysis of SOX4 and ITGAV deficient (CRISPR knockout) BT549 human and 4T1 murine TNBC cells as compared to control edited counterparts. Additionally, we performed SOX4 specific ChIP-seq studies in BT549 human TNBC cells. The objective of the study was to identify the molecular regulators and signaling pathways that mediate resistance to T cell mediated immunity. GSEA analysis of RNA-seq data showed that the 'interferon response' represented one of the top pathways for genes upregulated in SOX4 or ITGAV edited compared to control TNBC cells. In contrast, gene sets associated with TGF beta and TNF alpha/NF kappa b were negatively enriched in both Sox4 and Itgav edited TNBC cells. Further analysis of RNA-seq data showed that SOX4 or ITGAV edited TNBC cells contained higher mRNA levels of many interferon-stimulated genes (ISGs), including genes associated with important innate immune pathways such as RIG-I/MDA-5, cGAS - STING and the AIM2 inflammasome Overall design: Total RNA was extracted from control, SOX4 or ITGAV edited human BT549 and murine 4T1 cells cultured in complete RPMI media in biological triplicates. RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen # 74134) following the manufacturer's protocol. Total RNA was quality-checked using an Agilent BioAnalyzer 2000 instrument. RNA with an integrity number of greater than 9.5 was used for subsequent analyses. Total RNA was submitted to GeneWiz for RNA-seq analysis. Libraries were prepared with TruSeq RNA Sample Prep Kit v2 (Illumina). Library concentrations were quantified by Qubit (Invitrogen) and mixed equally for single-end 75bp sequencing using an Illumina NextSeq 500 instrument. Statistics for differentially expressed genes were calculated using DESeq2 (version 3.5) (47) and Cufflinks (48). Differential gene expression was analyzed using the DESeq2 (1.8.1) package in R using default settings (47). Principal component analyses were generated using the prcomp function in R and plotted with ggplot2. Human and mouse gene homologues were matched using the Mouse Genome Informatics annotation. Heatmaps were generated using the heatmap.2 function in R GSEA analysis of RNA-seq data showed that the 'interferon response' represented one of the top pathway for genes upregulated in SOX4 or ITGAV-edited compared to control murine 4T1 TNBC cells . Further analysis of RNA-seq data showed that Sox4 or Itgav edited 4T1 TNBC cells contained higher mRNA levels for of a large number ofmany interferon-stimulated genes (ISGs), including multiple genes associated with important innate immune pathways such as RIG-I/MDA-5, cGAS – STING and the AIM2 inflammasome Idenfication of DEG in Sox4 knockout 4T1 murine TNBC cells versus control cell counterparts Please note that each processed data file was generated from both replicates and is linked to the corresponding rep1 (*UT-1) sample records.