Transcriptomics, Regulatory Syntax, and Enhancer Identification in Heterogenous Populations of Mesoderm-Induced ESCs at Single-Cell Resolution

ESCs can adopt lineage-specific gene expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single cell-based assays were employed to catalogue gene expression, histone modifications, chromatin conformation and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further lineage specification. These assays identified cis-regulatory regions and transcription factors presiding gene expression programs occurring at defined ESC transitions and revealed the presence of heterogenous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs, and ESCs-derived cell lineages. Overall design: To catalogue gene expression, histone modifications, chromatin conformation and accessibility transitions in ESC populations and individual cells undergoing lineage specification. we induced ESCs differentiation and characterized their transcriptome and epigenome at specific developmental junctures. we examined genome wide , chromatin accessibilty and gene expression in both bulk and single cell in Naive ESC (Naive) (three replicates) and instructed ESC (Instructed), (two replicates). We characterized regulatory regions with active (H3K4me1 and H3K27ac, two replicates) epigenetic marks in Naive and instructed ESC. To gain insite on how chromatin accessibility remodelled and the transcriptionally heterogeneity of differntiated ESCs, we performed scATAC-seq and scRNAseq in ES-derived aPSM (aPSM) and multiomics in ES-derived HIFLR (HIFLR) . We then leveraged these datasets and, by combining chromatin accessibility, in situ Hi-C chromatin conformation in aPSM and HIFLR cells, genome editing, and reporter assays have identified regulatory regions directing initial Pax7 expression and activation of the myogenic and neurogenic programs. Please note that 'HIFLR_snATAC_fragments.tsv.gz' file was included on Dec 6, 2022.