Human ht 12 v4 beadchip arrays

Tumors were dissected free from stroma and mouse tissue, washed in PBS and immediately placed in RNAlater (Thermo Fisher Scientific). RNA was extracted using an RNeasy Mini Kit (Qiagen) and quality control was performed with the Agilent Bioanalyzer and RNA was quantified with the Qubit Fluorometer (Thermo Fisher Scientific) and stored at -80°C. Expression analysis was performed in triplicate with the Illumina HumanHT-12 v4 bead chip arrays (Illumina) and scanned on the BeadArray Reader (Illumia) according to the manufacturer's instructions. Raw data were normalized (quantile) and analyzed with Genome Studio software (Illumina). GSEA software was downloaded from the Broad website (http://www.broadinstitute.org/gsea/index.jsp). The number of permutations was 1,000 and the permutation type was gene_set. Gene expression microarray data has been submitted to the NCBI GEO repository as GSE90681.

RNA was extracted from whole blood in Tempus tubes using the MagMAX for Stabilized Blood Tubes RNA Isolation kit (Life Technologies) according to the manufacturer's instructions. Total RNA (2.5 μg) was then globin reduced using the GLOBINclear 96-well plate format kit (Life Technologies). The total RNA yield after globin reduction was determined in a NanoDrop 1,000 spectrophotometer (Fisher Scientific, Waltham, MA) and integrity assessed by Agilent 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples with RNA integrity number >6.0 were determined to be of acceptable quality for further analysis. Approximately 250 ng of globin-reduced RNA was used to prepare amplified and biotinylated antisense complementary RNA targets using the Illumina TotalPrep RNA amplification kit (Life Technologies). Seven hundred and fifty nanograms of labelled cRNA was hybridized overnight to Illumina human HT-12 v4 BeadChip arrays (Illumina Inc., San Diego, CA), which contained >47,000 probes (transcripts). Some genes were represented by more than one probe (transcript). The arrays were then washed, blocked, stained and scanned on an Illumina BeadStation 500. Signal intensity values from the scans were generated by the Illumina BeadStudio v2 software. All samples were randomized during all stages of processing to avoid any batch effect.