Non-disruptive 3D Profiling of Combinations of Epigenetic Marks in Single Cells

Recent advancements in single-cell sequencing and spatial omics technologies have enhanced our understanding of diverse cellular identities, compositions, and functions. However, the single-cell three-dimensional (3D) organization of the epigenome is still not well understood, due to an absence of spatial single-cell methods that allow high-resolution, locus-specific detection of combinations of epigenetic marks while maintaining the 3D organization of the genome. Here, we develop Epigenetic Proximity Hybridization Reaction (Epi-PHR), an image-based single-cell spatial epigenetic profiling technique. Epi-PHR enables high-sensitivity and high-resolution in situ detection of combinations of epigenetic marks at hundreds of single gene targets within the same individual cells, while retaining the 3D organization of the genome, a clear advantage over previous technologies. Phased Epi-PHR combined with chromatin tracing simultaneously detects allele-specific epigenetic states and chromatin conformations of an imprinting gene cluster in single cells, revealing associations between specific epigenetic mark enrichment and chromatin folding features for the distinct alleles from different parental origins. We expect Epi-PHR to be broadly applicable in research requiring single-cell spatial epigenetic information, and to help understand the combinatorial code of epigenetic marks and its relationship with chromatin folding in diverse biological and medical contexts. To interrogate the epigenetic state of imprinted loci, we cultured F1 Hybrid C57/CAST Trophoblast Stem Cells (TSCs). We then performed RNA-seq in triplicate to quantify allelic frequency of expressed genes. We then investigated whether H3K9me3 was deposited on paternal or maternal strands differentially using CUT&RUN.