Figure 4.
Joint scRNA-Seq and scATAC-Seq analysis of bottleneck populations. (A) UMAP of 1637 cells from scRNA-Seq and 1,186 cells from scATAC-Seq, aligned using Seurat algorithm with a custom defined gene-by-cell activity score matrix (see supplemental Methods). The number of HE cells was too few to be resolved by UMAP and was clustered with pre-HE. To gain enough statistical power for predicting E-P, we pooled reads from 10 nearest neighbors as “meta cells,” and paired scATAC meta cells to nearby scRNA meta cells. Additional details can be found in the supplemental Methods section. (B) UCSC genome browser tracks showing open chromatin signal of Cldn5 promoter and its predicted enhancers. Dots below each aggregated signal track represent signal from 50 sampled cells of each type. (C) Linear regression shows high correlation between Runx1 +23 enhancer chromatin accessibility and Runx1 expression levels (z-score transformed). Each point represents a paired ATAC-RNA meta cell in panel A, with pooled RNA expression on the y-axis and pooled enhancer accessibility on the x-axis. (D) Prediction of E-P interaction using linear regression. Predictions (points in blue shaded area, 5% of total candidate interactions) were made using P < .01 and regression coefficient >0.1. We recapitulated the majority of known E-P interactions that function during EHT, with the Runx1 +23 enhancer65 and Gfi1 enhancer66 among the top predictions. (E) TF binding patterns among called scATAC-Seq peaks assessed using chromVar,33 which defines a deviation score reflecting the accessibility change at binding sites of each TF across all cells. Binding sites were determined using DNA motif scan on the called enhancers, which does not discriminate TFs in the same family with very similar motifs. Top significant TFs based on Mann-Whitney U test are plotted for each stage. (F) ChromVar deviation score for selected TF motifs plotted on the UMAP, showing specific binding pattern for Tcf7 in Wnthi E, Sox17 in conflux E, Foxc2 in pre-HE, Gata2, and Klf2 in both pre-HE and IAC. Runx1 binding sites are highly accessible after bottleneck, but also exhibit medium to high level of chromatin accessibility in some early stage cells.

Joint scRNA-Seq and scATAC-Seq analysis of bottleneck populations. (A) UMAP of 1637 cells from scRNA-Seq and 1,186 cells from scATAC-Seq, aligned using Seurat algorithm with a custom defined gene-by-cell activity score matrix (see supplemental Methods). The number of HE cells was too few to be resolved by UMAP and was clustered with pre-HE. To gain enough statistical power for predicting E-P, we pooled reads from 10 nearest neighbors as “meta cells,” and paired scATAC meta cells to nearby scRNA meta cells. Additional details can be found in the supplemental Methods section. (B) UCSC genome browser tracks showing open chromatin signal of Cldn5 promoter and its predicted enhancers. Dots below each aggregated signal track represent signal from 50 sampled cells of each type. (C) Linear regression shows high correlation between Runx1 +23 enhancer chromatin accessibility and Runx1 expression levels (z-score transformed). Each point represents a paired ATAC-RNA meta cell in panel A, with pooled RNA expression on the y-axis and pooled enhancer accessibility on the x-axis. (D) Prediction of E-P interaction using linear regression. Predictions (points in blue shaded area, 5% of total candidate interactions) were made using P < .01 and regression coefficient >0.1. We recapitulated the majority of known E-P interactions that function during EHT, with the Runx1 +23 enhancer65  and Gfi1 enhancer66  among the top predictions. (E) TF binding patterns among called scATAC-Seq peaks assessed using chromVar,33  which defines a deviation score reflecting the accessibility change at binding sites of each TF across all cells. Binding sites were determined using DNA motif scan on the called enhancers, which does not discriminate TFs in the same family with very similar motifs. Top significant TFs based on Mann-Whitney U test are plotted for each stage. (F) ChromVar deviation score for selected TF motifs plotted on the UMAP, showing specific binding pattern for Tcf7 in Wnthi E, Sox17 in conflux E, Foxc2 in pre-HE, Gata2, and Klf2 in both pre-HE and IAC. Runx1 binding sites are highly accessible after bottleneck, but also exhibit medium to high level of chromatin accessibility in some early stage cells.

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