Comprehensive RNA-Seq Profiling of Tumor and Tissue

RNA was purified from tissue chunks of 3 primary tumors, 7 lung metastases, 8 normal lung and 12 normal mammary gland tissues (Supplementary Table 1) following the manufacturer’s protocol (PicoPure RNA Isolation Kit, ThermoFisher Scientific). Gene expression profiles were generated by mRNA-sequencing using an Illumina HiSeq 2000/2500. Briefly, mRNA libraries were made from total RNA using the Illumina TruSeq mRNA sample preparation kit and sequenced on an Illumina HiSeq 2000/2500 using a 2×50bp configuration with an average of 136 million read pairs per sample. Quality-control-passed reads were aligned to the mouse reference genome (GRCm38) using STAR^{80 (link), 81 (link)}. The alignment profile was determined by Picard Tools v1.64 (http://broadinstitute.github.io/picard/). Aligned reads were sorted and indexed using SAMtools and translated to transcriptome coordinates then filtered for indels, large inserts, and zero mapping quality using UBU v1.0 (https://github.com/mozack/ubu). Transcript abundance estimates for each sample were performed using SALMON^{82 (link)}. Raw SALMON read counts for all RNAseq samples were normalized to a fixed upper quartile.