RNA-Seq of Pt9: Tumor
RNA was extracted from frozen cell suspension using a Qiagen RNeasy Mini Kit. RNAseq libraries were prepared using an Illumina TruSeq Stranded mRNA Library Prep Kit. Total RNA was quantified using a Quant-iT RiboGreen RNA Assay Kit and normalized to 5 ng μ l−1. Sample was transferred into library preparation which was an automated variant of the Illumina TruSeq Stranded mRNA Sample Preparation Kit. This method preserved strand orientation of the RNA transcript. It used oligonucleotide dT beads to select messenger RNA (mRNA) from the total RNA sample. It was followed by heat fragmentation and complementary DNA (cDNA) synthesis from the RNA template. The resultant 500 bp cDNA then went through library preparation (end repair, base 'A' addition, adaptor ligation, and enrichment) using Broad-designed indexed adapters substituted in for multiplexing. The resulting libraries were quantified with qPCR using a KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. For Illumina sequencing, pooled libraries were normalized to 2 nM and denatured using 0.1 N NaOH before sequencing. Data were analysed using the Broad Picard Pipeline, which includes de-multiplexing and data aggregation.