This protocol describes the co-culture of cells from multiple species and, following RNA-Seq, the separation of reads according to species using the Sargasso bioinformatics pipeline in order to elucidate the effects of one cell type on the transcriptome of the others TWEET: Mixed species cell co-culture, RNA-Seq and read separation by species to elucidate non-cellautonomous transcriptional control. @UKDRI Hasel, P. et al. Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism. Nat Commun 8, 15132, (2017). Qiu, J. et al. Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons. eLife. 5, e20337 (2016).