Extended Data Fig. 9: Characterization of NDF_K161A stem cells and alternative normalization of nucleosome occupancy data.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Oct4 immunofluorescence in NDF_WT and NDF_K161A iPSCs. Scale bar = 5 μm. b, Vimentin immunofluorescence in undifferentiated iPSCs and differentiated EPIC-FB cells. Scale bar = 20 μm. c, Volcano plots showing differential gene expression in NDF-WT cells during differentiation. Left: iPSC vs cardiac progenitor cell (CPC) stage. Right: iPSC vs EPIC-derived fibroblast-like cell (EPIC-FB) stage. d, Volcano plots showing differential gene expression in NDF-K161A cells during differentiation. Left: iPSC vs CPC stage. Right: iPSC vs EPIC-FB stage. e, Volcano plot comparing gene expression between NDF-K161A and NDF-WT cells at the cardiac progenitor cell (CPC) stage. f, Volcano plot comparing gene expression between NDF-K161A and NDF-WT cells at the EPIC-FB stage. g, Boxplot of nascent RNA synthesis rates (TT-seq) in WT and NDF_K161A cells normalized to S. pombe spike-in nascent RNA. NDF_K161A shows reduced synthesis (n = 14,956 genes, P < 0.001, two-sided one-sample t-test, Box plots show median (centre), Q1 and Q3 (box), and min/max (whiskers).) h, Nascent RNA synthesis impairment (NDF_K161A/WT fold change) stratified by gene length quartiles (Q1-Q4, shortest to longest). Only genes with at least 1 read were analyzed. Shorter genes showed significantly greater impairment compared to longer genes (*P = 0.0266, ****P < 0.0001, two-sided t-test). (n = 9,498 genes with decreased nascent RNA levels in K161A cells relative to WT cells. Box plots show median (centre), Q1 and Q3 (box), and min/max (whiskers).) i, Metagene analysis of nucleosome positioning from MNase-seq in NDF_WT and NDF_K161A cells with S. pombe spike-in controls. Reduced nucleosome occupancy is observed in NDF_K161A. j, Metagene analysis of S. pombe spike-in nucleosome signals from the same MNase-seq experiment described in panel i. Analysis includes all S. pombe genes, demonstrating consistent digestion efficiency between NDF_WT and NDF_K161A samples. Genes with adjusted p-value (padj) < 0.05 and |log2 fold change | ≥ 1 from DESeq2 analysis are highlighted in red (c–f).
