The human transcription factor occupancy landscape viewed using high-resolution in situ base-conversion strand-specific single-molecule chromatin accessibility mapping

Chromatin accessibility profiling is a key tool for mapping the location of cis-regulatory elements (cREs) in the genome and tracking chromatin state dynamics during development, in response to various external and internal stimuli, and in disease contexts. Single-molecule footprinting (SMF) methods that rely on the labeling of individual accessible DNA bases have emerged in recent years as a powerful chromatin accessibility mapping approach, as they provide not just an average readout of accessibility over a given genomic position but also the distribution of accessibility states within a population at the level of individual original DNA molecules. However, methylation-based SMF approaches have often been limited either in their resolution or in labeling readout accuracy. We developed a deaminase-based high-resolution strand-specific single molecule footprinting approach that uses highly active sequence context-independent endogenous methylation-insensitive double-strand DNA (dsDNA) deaminases (CseDa01 and LbDa02 C[->]U/T-ssSMF), which convert accessible cytosines into uracils (which are converted to thymine after PCR amplification). We demonstrate the application of the method to mapping single-molecule accessibility states in human cell lines in both a short and a long-read format, and quantifying the occupancy states of individual transcription factors (TFs) as well as TF co-accessibility and strand-specific accessibility patterns.