RNA-seq analysis of tumor samples

RNA was isolated from tumor cell lines and mouse tumors with the Qiagen All Prep DNA/RNA kit. Quantification and quality assessments for RNA were performed with a Bioanalyzer (RNA integrity number >6.5; Agilent). Sequencing libraries were constructed with a TruSeq mRNA Library Preparation Kit using poly-A–enriched RNA (Illumina). The samples were run on a HiSeq 2500 Illumina sequencer generating 65-bp single-end reads. Raw sequence data and read counts data have been deposited in the Gene Expression Omnibus database (accession no. GSE145022). The sequence reads were mapped to the mouse genome (mm10) using TopHat (2.0.12). TopHat was run with default. Reads with mapping quality <10 and nonprimary alignments were discarded. Remaining reads were counted using HTSeq-count. Statistical analysis of the differential expression of genes was performed using DESeq2 (Love et al., 2014 (link)). Genes with false discovery rate for differential expression <0.01 were considered significant.