title: GSM6199332: C3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA from tumors was isolated using the MagMAX mirVana total RNA isolation Kit (Applied Biosystems). Following mechanical homogenization with an Ultra-turrax (T10 basis Ultra-Turrax, IKA), RNA was extracted according to the manufacturer's instructions and  stored at -80C until processed. RNA concentration was quantified using a Qubit 3.0 Fluorometer and its quality was examined in Agilent's 4200 TapeStation System 150ng of quality total RNA were used for the transcriptome interrogation using the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit according to the manufacturer's instructions (Illumina). Briefly, cytoplasmic and mitochondrial rRNAs as well as beta globin transcripts were depleted from the samples. The remaining RNA was fragmented and reverse-transcribed. A second strand cDNA synthesis step removed the RNA template while incorporating dUTP in place of dTTP in order to preserve strand specificity. Next, double-stranded cDNA was A-tailed, then ligated to Illumina anchors bearing T-overhangs. PCR-amplification of the library allowed the barcoding of the samples with 10bp dual indexes and the completion of Illumina sequences for cluster generation. Libraries were quantified with Qubit dsDNA HS Assay Kit and their profile was examined using Agilent's HS D1000 ScreenTape Assay. Sequencing was carried out in an Illumina NextSeq2000 using paired-end, dual-index sequencing (Rd1: 59 cycles; i7: 10 cycles; i5: 10 cycles Rd2:59 cycles) at a depth of 50 million reads per sample.