GSM6255185: IUE_PdgfraD482V, DNp53, FOXR2_Rep3_TP35; Mus musculus; RNA-Seq
Freshly isolated tumors were snap frozen and total RNA isolated using the NucleoSpin RNA kit. For isolation of polyA RNA, a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, E7490) was used for polyA RNA purification with a total of 1 μg good quality total RNA as input. The SMARTer Apollo NGS Library Prep System (Takara Bio, 640078) was used for automated polyA RNA isolation. For RNA sequencing library preparation, the library for RNA-seq was prepared by using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7760). After indexing via PCR enrichment (8 cycles), the amplified libraries together with the negative control were cleaned up for quality control analysis. To study differential gene expression, individually indexed and compatible libraries were proportionally pooled (~25 million reads per sample in general) for clustering in the cBot system (Illumina, SY-301-2002). Libraries at the final concentration of 15 pM were clustered onto a single-read flow cell using the TruSeq SR Cluster Kit v3 (Illumina, GD-401-3001), and sequenced to 51 bp using the TruSeq SBS Kit v3 (Illumina, FC-401-3001) on the Illumina HiSeq system.