mChIP-seq for multiplex epigenomic profiling uncovers cancer-specific histone features in cellular and circulating nucleosomes.

Epigenomic profiling elucidates regulatory roles of histone marks and provides a critical pathway for discovering non-invasive cell-free nucleosome epigenetic models. Here, we present multiplexed chromatin immunoprecipitation sequencing (mChIP-seq), a technology compatible with both cellular and cell-free samples for simultaneously profiling multifactorial epigenetic landscapes on multiple samples. By combining one-step sample indexing with a pool-and-split strategy, mChIP-seq enhances efficiency and reduces costs. Using mChIP-seq, we profiled H2A.Z and 10 histone modifications across cell lines representing nine cancer types, revealing a reduced correlation between H2A.Z and H3K4me3 at promoters in cancer cells compared to non-cancerous controls. We further extended mChIP-seq to establish cell-free mChIP-seq (cf-mChIP-seq) for mapping circulating cell-free nucleosomes. Profiling plasma samples for H2A.Z, H3K4me3, H3K27ac, and H3K9me3 revealed distinct histone-mark-associated cfDNA fragment patterns in breast cancer versus healthy controls, highlighting the potential of cf-mChIP-seq to expand liquid biopsy methodologies. Collectively, mChIP-seq offers a robust, scalable platform for epigenomic profiling in both cellular and cell-free contexts.