Extended Data Fig. 4: Application of scEpi2-seq in RPE-1 hTERT FUCCI cells.
a. Number of unique reads retrieved per single cell split by chromatin domain from wells containing a cell or a well left intentionally empty during sorting (n = 2 biological replicates). The boxplot is defined by the median ± interquartile range (IQR) and whiskers represent 1.5× IQR. b. Cells were quality-filtered based on the number of unique ChIC cut sites and DNA methylation. Top - Scatter plots showing the number of unique cut sites (x-axis, log10 scale) and per-cell 5mC levels (y-axis) in the RPE-1 data set. Similar to the top panel, but zoomed in showing only cells with higher numbers of unique ChiC counts ( > 8000 reads). Dots are colored by fraction reads in peaks (FRiP). c. Violin plot showing the fraction of reads in peaks (FRiP) for QC-filtered cells in the RPE-1 hTERT FUCCI data set (n = 2 biological replicates). Data are grouped for different modifications assessed with scEpi2-seq. The boxplot is defined by the median ± interquartile range (IQR) and whiskers represent 1.5× IQR. d. Normalized density plot showing the number of CpGs detected per single RPE-1 hTERT FUCCI cells (x-axis, log10 scale stratified by histone mark (columns) and QC status (rows). e. Distribution of RPE-1 methylation levels (WGBS) within peaks called from scEpi2-seq data (top row). Also included are methylation levels for random regions with the same size distribution (bottom row). f. Heatmap with histone modification signal intensities (number of ChIC counts) of single RPE-1 cells (rows) for a representative region on chromosome 2. The genomic region is equivalent to the one presented in Fig. 1b. g. Single-cell heatmap for cut site spacing in RPE-1 cells for the 250 cells with highest unique coverage per chromatin mark and arranged by the unique read depth. The read pair correlation shows characteristic oscillations related to nucleosome spacing. h. Comparison of in silico bulk aggregates of relative methylation extracted from scEpi2-seq data and WGBS data of the same cell line in 25 kb bins across the genome. Correlation estimation was performed using Pearson correlation test with a correlation coefficent of 0.9 and the and a p-value < 2.2×10−16 i. Average CpG methylation in relation to distance from the cut site. Similar to K562 data, H3K36me3-decorated, actively transcribed regions show the highest average methylation. j. Coverage plot of Signac normalized pseudobulk H3K27me3, H3K36me3, and H3K9me3 signal in RPE-1 hTERT FUCCI cells for a region containing the FTH gene (highly expressed in RPE-1 hTERT cells).
