GSM2731746: CC-223 Mouse #231; Homo sapiens; RNA-Seq
Please refer to the manuscript by Orr-Asman et al for detailed description of the extraction protocol. Briefly, RNA was isolated from each ~50 mg of each tumor sample using the mirVana miRNA Isolation Kit (ThermoFisher) according to the manufacturer protocol. Target RNA enrichment was performed from ~750 ng of RNA by isolation of polyA RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). PrepX PolyA script was run for automated polyA RNA isolation. RNA-seq library preparation was done using NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs) with dUTP for cDNA synthesis to maintain strand specificity, indexing via 11 cycles of PCR enrichment and finally cleaning up of the amplified library by AMPure XP beads for QC analysis. The quality and yield of the purified library was analyzed with a Bioanalyzer (Agilent) and a DNA high sensitivity chip; then the concentration was quantified by qPCR using the Kapa Library Quantification kit (Kapabiosystem). Individually indexed and compatible libraries were proportionally pooled (~25x106 million reads per sample) for clustering with a cBot system (Illumina). Libraries at the final concentration of 15 pM were clustered onto a single read flow cell using Illumina TruSeq SR Cluster kit v3, and sequenced to 50 bp using TruSeq SBS kit on an Illumina HiSeq system.
GEO Accession
GSM2731746