Single cell RNA-seq in the sea urchin embryo show marked cell-type specificity in the Delta/Notch pathway

Overall, we conclude that single cell RNA-seq analysis in this embryo is revealing of the cell types present during development, of the changes in the gene regulatory network resulting from inhibition of various signaling pathways, and of the selectivity of these pathways in influencing developmental trajectories. Overall design: Adult S. purpuratus were obtained from South Coast Bio-Marine [e-mail] and were spawned using 0.5?M KCl injections intracoelomically. DAPT (Tocris) was added at fertilization to 8?µM, a concentration previously determined to effectively perturb Delta/Notch signaling without affecting overall development (Materna & Davidson, 2012). C59 (Cellagen) was added at 0.5?µM at fertilization as previously described (Cui et al., 2014). Embryos were cultured and, at appropriate developmental stages, were dissociated into single cells as described (McClay, 2004). We started with 50?ml cultures containing 0.25% of embryos (125?ul of embryos in 50?ml). The full volume was dissociated into single cells. We obtained 12 million single cells from which 5,000 were used for the DropSeq.The single-cell RNA-seq protocol was performed using the ChromiumTM Single Cell 3' reagent kit v2 chemistry and cells were loaded on a GemCode Single Cell Instrument (10× Genomics, Pleasanton, CA).