RNA-seq of AOM-DSS-induced Colon Tumors

RNA from AOM-DSS-induced macrodisected tumors was isolated using TRIzol Reagent (Thermo Fisher Scientific, 15596018) and purified using the RNeasy® Protect Mini Kit (Quiagen, 74127) according to the manufacturer’s manual. RNA-sequencing was performed at the Core Facilities of the Medical University of Vienna. Sequencing libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina according to manufacturer’s protocols (New England Biolabs). Libraries were QC-checked on a Bioanalyzer 2100 (Agilent) using a High Sensitivity DNA Kit for correct insert size and quantified using Qubit dsDNA HS Assay (Invitrogen). Pooled libraries were sequenced on a NextSeq500 instrument (Illumina) in 1x75bp single-end sequencing mode. Approximately 25 million reads were generated per sample. Reads in fastq format were aligned to the mouse reference genome version GRCh38⁷³ with Gencode mV23 annotations⁷⁴ using STAR aligner^{75 (link)} version 2.6.1a in 2-pass mode. Reads per gene were counted by STAR, and differential gene expression was calculated using DESeq2^{76 (link)} version 1.20.0. and analyzed using the Gene Set Enrichment Analysis (GSEA) tool provided by the Broad Institute.^{39 (link),40 (link)}