Fig. 1: epi-gSCAR workflow schematics and methylation readout for the DLX4 locus.

a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.