3D epigenome of glial cell types in developing human cortex

Key Points:

• Researchers isolated and fixed cells from the developing human cortex (gestational weeks 15–24) for downstream analyses including FACS, RNA-seq, ATAC-seq, WGBS, and PLAC-seq, maintaining procedures at low temperatures to preserve sample integrity.
• Multi-omics approaches were employed: RNA-seq libraries were prepared from sorted cells to analyze gene expression; ATAC-seq assessed chromatin accessibility; whole-genome bisulfite sequencing (WGBS) profiled DNA methylation; and PLAC-seq mapped chromatin interactions mediated by H3K4me3.
• Computational analyses included cell-type deconvolution with CIBERSORTx, motif enrichment with HOMER, transcription factor footprinting via TOBIAS, and machine learning models (GKM-SVM) to predict chromatin accessibility and variant effects; these tools helped identify cell-type-specific regulatory elements and variant impacts.
• Functional validation involved CRISPR interference targeting human accelerated regions (HARs) and luciferase reporter assays comparing human and chimpanzee HAR variants, as well as in vitro culture and single-cell RNA-seq of outer radial glia (oRG) to study gene regulatory effects.
• Ethical compliance was ensured with appropriate approvals for human prenatal tissue use and animal experiments, adhering to institutional guidelines and regulatory standards.