RNA-seq of co-culture experiment between human HUVEC endothelial cells and mouse glioma GL126 cells

Increased awareness for the role for angiocrine factors in stem cell biology, in general, has suggested a role for angiocrine factors in the maintenance of GSC stemness and also there are reciprocal bi-directional signals from endothelial cells (ECs) to glioma (Gl261) and vice versa. To extend, refine and elucidate perfusion-independent modes of GBM-EC communication (i.e., distinguishing contact dependent or contact independent routes), we carried-out the following experiments: ECs from umbilical veins tagged with RFP (HUVEC-RFP) were cultured with mouse glioma line Gl261 fluorescently labelled with GFP (Gl261-GFP) until reaching confluency by 72 h post culturing. Cells were than harvested, RNA extracted, and the combined co-culture transcriptomes were sequenced from a generated cDNA library without prior separation of the respective cell types (sample named as Co). The Co transcriptome was compared to that of an artificial mixtures of the two cell types (Gl261-GFP and HUVEC-RFP) in the exact cells ratio attained by the end of the co-cluttering aided by FACS-based determination of the GFP/RFP ratio (Gl261:HUVEC - 3:1) attained by the end of the co-cluttering aided by FACS-based determination of the GFP/RFP ratio (sample named as Sep). In this procedure advantage was taken on the different species from which GBM and EC are derived and specialized bioinformatics tools allowing to unambiguously assign the transcripts to their respective GBM (mouse) or EC (human) origins, thereby circumventing confounding factors associated with cell purification