Fig. 1: Flowchart of experimental design.
From: The regulatory architecture of the primed pluripotent cell state
a Interactome construction. Two independent EpiSC lines were treated with 5 differentiation conditions and 33 small molecule perturbations, generating 276 expression profiles. These profiles served as input for the ARACNe algorithm to create the “first-generation” EpiSC interactome. b Inference of candidate MRs. 144 expression profiles from 5 distinct lineage-specific differentiation time courses were used to generate signatures. The VIPER algorithm then analyzed these signatures to identify candidate MRs. c Validation of candidate MRs. Two independent screens investigated whether silencing of candidate MRs could modulate the pluripotent state, resulting in 132 validated MRs. The first screen examined whether candidate MR silencing altered endogenous Oct4 protein levels, while the second screen used PLATE-seq to determine if candidate MR silencing recapitulated the EpiSC differentiation signature. d Functional validation of MRs. 70 selected MRs underwent CRISPR-mediated knockout to investigate pluripotency defects through cell culture assays and teratoma formation. e Network modularity analysis. PLATE-seq expression profiles from MR silencing were used to assemble a comprehensive, experimentally-derived regulatory network. Four distinct MR communities were identified within this network. f Regulatory architecture inference. Network hierarchy and centrality were analyzed, followed by further examination of community structure.
