Illumina HiSeq 4000 paired end sequencing; RNA-seq of human ex vivo ovarian cancer models with matched stromal cells - part II
RNA-seq of human ex vivo ovarian cancer models with matched stromal cells - part II
Ascites was obtained via the Manchester Cancer Research Centre Biobank (Study number: 16_STTA_01). Solid tumour samples were obtained via the Manchester Cancer Research Centre Biobank (Study number: 16_STTA_01). The acsites fluid was centrifuged at 500xg for 10mins at 4oC and cell pellets pooled in HBSS. Red blood cells were removed using a red blood cell lysis buffer (Miltenyi Biotec) as per the manufacturer's instructions. Tumour cells were counted and plated at 1 million cells in two 75cm2 Primaria flasks, one containing OCMI media and the second containing a 50:50 OCMI media: ascitic fluid mix. Chemo naïve samples were plated onto collagen-coated 75cm2 flasks and cultured in OCMI media containing 5% Hyclone FBS.Solid tumours were processed using a tumour dissociation kit (Miltenyi Biotec) following the manufacture's instructions. In brief, samples were dissected into small pieces (2-4mm), transferred to a gentleMACS C tube containing RPMI media supplemented with enzymes H, R and A. Tubes were loaded on to the GentleMACS dissociator and the pre-programmed “h_tumour_01” was initiated. Once completed, the sample was incubated at 37oC with continuous, gentle rotation. This dissociation and incubation process was repeated two additional times. Samples were then filtered using a MACS SmartStrainer (Miltenyi Biotec), centrifuged at 300xg for 7 minutes and plated onto collagen-coated 12.5cm2 flask. For both Cultures were incubated for 2-4 days at 37oC in a humidified 5% CO2 and 5% O2 atmosphere. Media was replaced every 3-4 days. Upon cell attachment, stromal cells were separated from the mixed sample using 0.05% trypsin-EDTA, and plated in gelatin-coated flasks in OCMI media containing 5% FBS. Once tumour cells reach 95% confluency, cells were passaged using 0.25% Trypsin-EDTA, centrifuged in DMEM containing 20% FBS and replated at a 1:2 ratio. For long term storage, cells were frozen in Bambanker. RNA was extracted using RNeasy Plus Mini kit (Qiagen) from two 75cm2 flasks, quantified using a Qubit fluorometer (Life Technologies). cDNA libraries were prepared following the TruSeq Stranded mRNA sample preparation protocol (Illumina).
Experimental Factor: sampling site
stromal cells
Experimental Factor: cell type
fibroblast