A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

Empirical optimization of stem cell differentiation protocols is time consuming, is labor intensive, and typi- cally does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet sin- gle-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA ‘‘barcodelets’’ to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combina- torial effects of signaling pathway activation on gene expression. We further develop a data-driven frame- work for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differen- tiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigatingQ2 and understanding how cooperative signaling pathways drive cell fate acquisition.