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

Transcription factors (TFs) regulate gene expression by binding to cis-regulatory elements in the genome. Understanding transcriptional regulation requires genome-wide characterization of TF occupancy across different chromatin contexts, yet simultaneous assessment of TF binding for multiple factors remains technically challenging. Here, we describe a detailed and reproducible protocol for cFOOT-seq, a cytosine deaminase-based genomic footprinting assay by sequencing, which enables antibody-independent, base-resolution profiling of chromatin accessibility, nucleosome organization, and TF occupancy. In cFOOT-seq, the double-stranded DNA (dsDNA) cytosine deaminase SsdA_tox 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.