Fig. 1: Ectopic ICR sequences recapitulate chromatin states of endogenous ICRs.

a, Experimental overview of stable cell line generation with methylated or unmethylated donor plasmids using RMCE. b, Tabular summary of methylation analysis for all integrated ICRs, control DMR and promoter sequences. Endogenous methylation (Endog. meth.) describes the methylation state of the endogenous locus in mESCs. Mat., maternal methylation; Pat., paternal methylation; n/a, no methylation. Size, CpG density (in 100 bp) and GC content (%) are indicated. Total methylation percentages of DNA sequences integrated via RMCE measured by bsPCR is shown for experiments using unmethylated (− M.SssI) and premethylated (+ M.SssI) donor plasmids. n/d, not determined. Asterisks indicate measurements obtained from Lienert et al.³. c, Detailed methylation analysis for the ectopic Airn ICR. CpG positions within the Airn ICR sequence are indicated with black vertical lines. Amplified regions for bsPCR are depicted, and single-molecule measurements are shown as black circles corresponding to methylated CpG dinucleotides and white circles to unmethylated CpG dinucleotides. CpG positions marked with ‘x’ correspond to unaligned nucleotides due to sequencing errors. Aggregated methylation values are displayed as color-coded vertical lines at the respective CpG position. d, ChIP-qPCR measurements at ectopic and endogenous ICRs compared to an intergenic site. H3K9me3, blue; H3K4me2, orange. Data points indicate individual technical replicates.