T_501_Hom_S_3_p
5 million melanoma cells were injected subcutaneously on the back of CD1 nude female mice. Tumours were grown to 1000mm3, following which animals were culled by overdoes of anaesthesia following cardiac puncture and tumours were harvested. For isolation of cells from whole tumours, the tumours were cross chopped with a scalpel as small as possible and then dissociated with pre-warmed Liberase (Roche) at 50ug/ml inserum free media in a humidified incubator at 37oC for 120mins. Serum containing mediawas then added, to neutralise the enzymes, and the samples were centrifuged and resuspendedin PBS for FACS sorting.     Blood was removed by cardiac puncture while mice were under anesthesia for isolating circulatory tumour cells (CTCs). The blood was then diluted in PBS and separated by density gradient centrifugation using Ficoll-Paque (GE Healthcare). The plasma and PBMC layer was then carefully removed and diluted in PBS for FACS sorting.    Cell sorting from tumours was performed using a BD Biosciences FACS Aria Fusion with the excitation laser 488nm and the emission filter 530/30nm bandpass for GFP and excitationlaser 561nm and the emission filter 610/20nm bandpass for mCherry. The gating was set asfollows: FCS-H/FSC-A was used to exclude any events that could represent more than 1 celland FSC/SSC were used to gate out debris based on size. GFP/mCHerry was used to selectGFP or mCherry positive cells. Immediately after FACS sorting sampleswere centrifuged, PBS removed and the cells were thoroughly resuspended in Qiazol.  RNA was extracted using an Rneasy Plus Micro Kit (Qiagen).  Strand-specific RNA-seq libraries were prepared using the Illumina workflow with the TruSeq® Stranded mRNA Sample Preparation Kit.