RNA-Seq of Pt4: 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. The mRNA enrichment step was omitted before library preparation. 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.