We developed a human postmortem brain-applicable ‘Fiber-seq' protocol for genome-scale, PCR-free single molecule sequencing of on average 8-10kb long chromatin fibers, to map nucleosome positioning and nucleosome-depleted regions which include, for example, active promoters and enhancers. We generated two Fiber-seq reference sets, one from sorted non-neuronal NeuN- prefrontal cortex (PFC) nuclei from a 32 year old female donor, with total of 11,143,795 reads/fibers, and median of 30-fold genomic coverage. The other reference set was generated on sorted prefrontal NeuN+ neurons, from two 24 year old brain donors (1 female/1 male), with a combined total read/fiber count of 4,749,556 and median of 20-fold genomic coverage. Each of the peaks in the Fiber-seq libraries seq signal correlated with the ATAC peak levels in the neuronal and non-neuronal ATAC-seq libraries, respectively (promoters, clusters 1, 3; NeuN+, r = 0.574; NeuN-, r=0.683, P&#x3C;0.00001; enhancers clusters 2, 4; NeuN+, r = 0.41; NeuN-, r=0.55, P&#x3C;0.00001). NeuN+ versus NeuN- Fiber-seq peak differentials showed extremely strong correlations with corresponding cell type specific difference in ATAC-seq dataset. For NeuN+ enhancers, R-squared value of 0.85 with a p-value of 0, for NeuN- enhancers, R-squared value of 0.98 with a p-value of 0. Therefore, enhancer-associated nucleosome depleted regions as mapped in our long-read single molecule chromatin fiber assay shows excellent overlap with open chromatin regions as defined by conventional short-read sequencing of transposase-digested chromatin. However, Fiber-seq detected 19,978 highly significant peaks which, while showing some non-significant/near-background ATAC-Seq alignments, lacked a corresponding site-specific enrichment in conventional short-read ATAC- and ChIP-seq datasets. However, 60% of these peaks were in repetitive DNA elements. Therefore, long read single chromatin fiber sequencing, which exceed the base pair length of conventional short-read libraries by two orders of magnitude, could capture many actively regulated repeat elements at the site of specific gene loci, that otherwise would remain unmappable by conventional ATAC-, and histone- ChiP-seq.

Single Chromatin Fiber Profiling in the Human Brain