Nucleosomes are the basic unit of chromatin, but genome-scale maps of nucleosome positioning along chromatin fibers do not exist for brain and many other complex tissues. Conventional chromatin accessibility assays designed to map nucleosome-depleted regions via nucleolytic digestion face major limitations such as limited resolution and sequence bias, with additional shortcomings from PCR-generated short-read libraries including poor annotation for an estimated 50% of the human genome. To address this, we tested a brain-adapted single-molecule chromatin fiber sequencing (Fiber-seq) protocol designed for amplification-free adenine-methyltransferase tagging of extranucleosomal DNA in neuronal and, separately, non-neuronal nuclei in situ.

We apply a single-molecule chromatin fiber sequencing (Fiber-seq) protocol designed for amplification-free cell-type-specific mapping of the regulatory architecture at nucleosome resolution along extended ∼10-kb chromatin fibers to neuronal and non-neuronal nuclei sorted from human brain tissue. Specifically, application of this method enables the resolution of cell-selective promoter and enhancer architectures on single fibers, including transcription factor footprinting and position mapping, with sequence-specific fixation of nucleosome arrays flanking transcription start sites and regulatory motifs. We uncover haplotype-specific chromatin patterns, multiple regulatory elements cis-aligned on individual fibers, and accessible chromatin at 20,000 unique sites encompassing retrotransposons and other repeat sequences hitherto “unmappable” by short-read epigenomic sequencing. Overall, we show that Fiber-seq is applicable to human brain tissue, offering sharp demarcation of nucleosome-depleted regions at sites of open chromatin in conjunction with multi-kilobase nucleosomal positioning at single-fiber resolution on a genome-wide scale.