Fig. 4

scNMT-seq of Tet-TKO cells reveals impaired DNA demethylation of erythroid enhancers during primitive erythro- poiesis. a Schematic summarising the scNMT-seq chimaera assay. Fluorescently labelled Tet-TKO ESCs are injected into wild type blastocysts, transferred into pseudopregnant hosts then collected at E8.5. FACS is used to isolate specific populations (CD41+, erythroid; KDR+, Haematoendothelial progenitors; CD41+ KDR+, blood progenitors and CD41−, KDR−) of both labelled KO cells (red) and non-labelled WT host cells (blue) which are processed and sequenced using scNMT-seq. b Scatter plot displaying expression levels of the haemoglobin alpha adult chain 1 gene (Hba-a1) in cells ordered along a reconstructed primitive erythropoiesis trajectory. Cells are coloured by genotype, WT (N=301, top) and Tet-TKO (N=221, bottom). The line displays the LOESS curve. c As b for Dnmt and Tet genes, and Uhrf1. To avoid cluttering the LOESS curves are shown without the corresponding data points. d Scatterplot displaying global CpG methylation in cells ordered along the same pseudotime trajectory as b and coloured by genotype. The line displays the LOESS curve. e DNA methylation (yellow) and chromatin accessibility (green) profiles quantified over multiple genomic contexts in WT (N=67,top) and Tet-TKO (N=57, bottom) erythroid cells. Each column corresponds to a different genomic context: promoters (N=18,329), surface ectoderm enhancers (N=2138), haematoendothelial progenitors enhancers (N=3616), and erythroid enhancers (N=4319). Shown is the mean +/− 1 standard deviation in running averages of 50bp windows around the centre of the genomic annotation (2kb upstream and downstream). f Boxplots showing the distribution of DNA methylation (top) and chromatin accessibility (bottom) in erythroid cells in WT (N=67, blue) and Tet-TKO (N=57, red) at different genomic annotations