FENGC: Targeted, Multiplexed, and Integrated Single-Molecule Profiling of Chromatin Accessibility and DNA Methylation.

DNA methyltransferase accessibility protocol for individual templates (MAPit) enables localization of DNA-protein interactions by probing the native chromatin architecture in nuclei or permeabilized cells using exogenously applied DNA methyltransferases. By preserving the integrity of DNA in chromatin, MAPit enables simultaneous mapping of nucleosome positioning, transcription factor binding, and endogenous CpG methylation in a single assay-while maintaining linkage between regulatory modules along chromatin fibers. Although MAPit and other sequencing-based approaches can be applied genome-wide, there is a growing demand for orthogonal approaches that validate target discovery and offer deeper molecular resolution at defined loci. Here, we describe a detailed protocol for flap-enabled next-generation capture (FENGC), a cost-effective method for targeted, multiplexed enrichment of DNA sequences for epigenetic and genetic analysis. FENGC precisely excises and enriches tens to hundreds of user-defined targets, generating sequencing-ready libraries with superior on-target rates compared to existing methods, all at a fraction of the cost. When paired with MAPit, the MAPit-FENGC platform enables single-molecule resolution of CpG methylation patterns in the context of chromatin architecture and transcription factor occupancy.