TRAP seq in 3D angiogenesis assays reveals a distinct endothelial translatome associated with early and late stages of morphogenesis.

Endothelial cells (ECs) are often a minority cell type in a tissue, limiting the utility of bulk sequencing approaches. Single cell sequencing lacks sensitivity and requires disruptive tissue digestion techniques. TRAP seq (Translating Ribosome Affinity Purification) or "RiboTag" has been used to overcome these limitations. Multicellular co-culture systems allow primary ECs to differentiate and undergo tubular morphogenesis in cell culture, however similar limitations exist with these in vitro assays, as ECs are under-represented by as much as a factor of 10 in many assays. We sought to use TRAP seq to better understand the gene expression landscapes that drive these morphogenic events. We found TRAP seq selectively enriches for endothelial RNA in two distinct co-culture paradigms, in both the planar and fibrin bead co-culture assays. Intriguingly, the use of this technology in vitro, revealed distinct gene expression changes in blood vessel development and in the mitotic cell cycle, with genes unique to early and late phases of morphogenesis. It is widely accepted that expression of the NOTCH signaling pathway is a regulator of angiogenesis in vivo. Correspondingly, we found that many NOTCH related genes relevant to endothelial cell biology, were changing across this morphogenic process, underpinning the importance and utility of this technology in 3D multicellular cultures that model in vivo environments.