A Cytosine Deaminase–Based Genomic Footprinting Assay (cFOOT-seq) for Detecting Transcription Factor Occupancy

converts cytosine to uracil in accessible chromatin, whereas TF binding and nucleosome occupancy locally protect DNA from deamination. Using the FootTrack analysis framework, deamination patterns generated by cFOOT-seq are quantitatively analyzed to derive standardized footprint and chromatin organization profiles at base resolution across the genome. Because cFOOT-seq preserves genomic DNA integrity during deamination-based footprinting, it is compatible with ATAC-seq-based chromatin enrichment. ATAC-combined implementations reduce sequencing depth requirements and improve scalability for footprint-focused analyses, supporting applications in low-input and single-cell settings. This protocol provides a practical framework for genome-wide TF footprint profiling and can be readily applied to dissect gene regulatory mechanisms in development, immunity, and disease, including cancer. Key features • cFOOT-seq provides antibody-independent, base-resolution, genome-wide profiling of TF occupancy together with chromatin accessibility and nucleosome organization in a single assay. • Using the FootTrack analysis framework, cFOOT-seq enables quantitative and comparative analysis of TF footprint patterns and occupancy across the genome. • cFOOT-seq preserves genomic DNA integrity during deamination-based footprinting and is compatible with ATAC-seq-based chromatin enrichment. • Integrated with ATAC, cFOOT-seq enables sensitive and in-depth TF footprint analyses at low sequencing cost, supporting single-cell applications.