Comparative analyses of ChIP-seq, CUT&RUN and CUT&Tag for Polycomb chromatin profiling

Chromatin profiling methods such as ChIP-seq, CUT&RUN, and CUT&Tag differ substantially in background structure, signal distribution, and resolution, complicating direct quantitative comparison across platforms. In this study, we systematically compared conventional and double-crosslink ChIP-seq, CUT&RUN, and CUT&Tag by profiling the Polycomb-associated histone modification H3K27me3 in human cardiomyocytes and the PRC2 catalytic subunit EZH2 in pluripotent stem cells. To enable cross-assay comparison, we developed a biologically informed normalization strategy based on stable Polycomb reference loci, allowing harmonization of signal scales while preserving assay-intrinsic signal architecture. This approach revealed CUT&RUN to preferentially capture broad H3K27me3 domains, whereas CUT&Tag provides sharper and more localized enrichment for both H3K27me3 and EZH2. Together, our results establish a practical framework for cross-platform epigenomic comparison and guide the selection of chromatin profiling strategies.