Time series scRNAseq analysis in mouse and human informs optimization of rapid astrocyte differentiation protocols

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence in the brain and spinal cord. Here we used single-cell RNA sequencing (scRNAseq) to analyze over 298,000 cells and nuclei during macroglia differentiation from mouse embryonic and human induced pluripotent stem cells. We computationally identify candidate genes involved in fate specification of glia in both species, and report heterogeneous expression of astrocyte surface markers across dif- ferentiating cells. We then used our scRNAseq data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multiomic, dual single nuclei (sn)RNAseq/snA- TACseq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets enable future optimization of glial differentiation protocols and provide insight into human glial differentiation. Overall, single-cell RNA and ATAC sequencing of time points during glial differentiation in human and mouse stem cells. Some time points were replicated as discussed in manuscript. Multiomic (dual snRNAseq + snATACseq) analysis of mouse astrocytes given either BMP4/FGF1 or CNTF growth factor treatments for 5,7, or 9 days total.