Extended Data Fig. 4: Global DNA methylation and transcriptome reprogramming of mESCs transitioned from SL to different media conditions after 48 hours.
(a) Principal component analysis (PCA) of genome-wide DNA methylation and DNA maintenance methylation levels of mESCs grown in different culture conditions for 48 hours. M-M-Dyad-seq was performed on three independent replicates for each culture condition. (b) Loss of DNA methylation after culturing mESCs in 2iL conditions for 48 hours is associated with a reduction in 5mCpG maintenance levels. Each point represents genomic tilling of 100 kb. (c) Genome-wide 5mCpG levels quantified using H-M-Dyad-seq for mESCs grown under different conditions. The violin plots are made over 2,599 genomic bins. (d) Genome-wide 5hmCpG levels quantified using H-H-Dyad-seq for mESCs grown under different conditions. The violin plots are made over 2,605 genomic bins. (e) The first two principal components show distinct transcriptomes of mESCs grown under different conditions. Bulk RNA-seq was performed in triplicate. (f) Heatmap of the expression level of genes related to de novo methylation, maintenance methylation, and demethylation pathways. (g,h) Gene pathway enrichment analysis for differentially expressed genes (hypergeometric test with Benjamini-Hochberg correction) was performed using Metascape33. Panel (g) shows gene sets associated with specific pathways that are highly expressed in 2iL and ML conditions, lowly expressed in No, and not differentially expressed across SL, BL, and GL conditions. Panel (h) shows gene sets associated with specific pathways that are highly expressed in the No condition, lowly expressed in 2iL and ML, and not differentially expressed across SL, BL, and GL.
