Abstract
The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues.
Highlights
Distal Intronic Exonic PromoterDistal Peaks MoƟf P-value CTCF 1E-260 BORIS 1E-179 Sp1 1E-80 E2F7 1E-69 E2F1 1E-68as compared to distal elements, implying that size selection for the subnucleosomal fraction could lead to an over-representation of transcription start sites (TSS) in ATAC-seq peaks
The nucleosome-free regions (NFR) that overlapped promoters were most significantly enriched for the binding motif of Sp1, a transcription factor involved in many cellular processes, and that of ETS factors, which are implicated in a wide variety of functions via transcriptional regulation
Distal NFR were most significantly enriched for the CTCF binding motif, suggesting that many of these peaks are likely involved in the 3-D organization of the genome, since CTCF has been implicated in long-range chromatin interactions, such as those between enhancers and their targets[13]
Summary
As compared to distal elements, implying that size selection for the subnucleosomal fraction could lead to an over-representation of TSS in ATAC-seq peaks. In contrast to this expectation, only a third of NFR found in both 3-d-200K and 3-e-200K corresponded to promoters (2 kb upstream of TSS), and nearly half fell in intergenic regions (Fig. 5). Distal NFR were most significantly enriched for the CTCF binding motif, suggesting that many of these peaks are likely involved in the 3-D organization of the genome, since CTCF has been implicated in long-range chromatin interactions, such as those between enhancers and their targets[13]. Modified ATAC-seq generates high quality chromatin accessibility data for pig lung, muscle and spleen. Tissue replicates clustered together based on read alignments (Fig. 7a), and NFR were consistent between biological replicates, with 68%, 87%, and 64% of peaks detected in both replicates of lung, muscle, and spleen, respectively
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