An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues

Abstract

We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.

Figure 1

Comparison of the Omni-ATAC protocol to standard ATAC-seq or Fast-ATAC. (a) Heat-map-based representation of ATAC-seq quality control metrics, including estimated library size (“Lib size”; purple), percentage of reads mapping to mitochondrial DNA (“% mito”; blue), and enrichment of signal at TSSs (orange) (Online Methods). Deeper color is used to depict the most desirable value of each statistic and ranges following a linear scale starting at 0 (white) and ending at the maximum value for the given cell type. In the case of the percentage of reads that map to mitochondrial DNA (% mito), the color scale starts at the maximum value (white) and ends at 5% (blue). All values were determined from 5 million random aligned reads. Two technical replicates are shown per sample as numeric values within each box. (b) Fraction of reads in peaks that map to TSSs (±500 bp of TSS) and distal elements (>500 bp from TSS) from libraries generated in this study, using the standard ATAC-seq method (“Standard”), the Fast-ATAC-seq method (“Fast”), or the Omni-ATAC protocol (“Omni”). Each bar represents a single technical replicate. **P < 0.01; ***P < 0.001 by two-tailed unpaired Student’s t-test comparing the fraction of reads in peaks to reads outside of peaks. All values were determined from 5 million random aligned de-duplicated reads. mESC, mouse embryonic stem cell. (c) Overlap of GM12878 peaks called from 60 million reads using standard ATAC-seq, DNase-seq, and Omni-ATAC. All input reads were trimmed to equal lengths (36 bp) before alignment. Data represent the mean of three individual downsampling replicates. (d) Heat-map-based representation of ATAC-seq quality control metrics, including estimated library size, percentage of reads mapping to mitochondrial DNA, and enrichment of signal at TSSs, as shown in a. Deeper color is used to depict the most desirable value of each statistic. All values were determined from 5 million random aligned reads. Two technical replicates are shown per sample.

Figure 2

Omni-ATAC in defined regions of the post-mortem human brain. (a) Pearson correlation heat map showing sample-by-sample unsupervised clustering on all peaks identified across all regions. Biological donor and brain region are indicated by the color across the top. CC, corpus callosum; CN, caudate nucleus; CB, cerebellum; HIP, hippocampus; MFG, middle frontal gyrus. All regions are represented by four technical replicates per donor. (b) Significance of enrichment of disease-specific GWAS polymorphisms in the uniquely accessible regions of five different brain regions (shown in the top left image) from individuals with the indicated diseases. The empirical P value is depicted colorimetrically with reference to association-based permutations of the GWAS SNPs. Nonsignificant empirical P values are represented in white. (c) Schematic of the experimental strategy for the generation of chromatin accessibility profiles from 50-μm frozen tissue sections with adjacent relevant histological staining. (d) Pearson correlation heat map showing sample-by-sample unsupervised clustering on all peaks. Data derived from the 20-mg tissue fragments represent the mean of four technical replicates from a single biological donor. Data derived from the 50-μm tissue sections show individual technical replicates. (e) Representative images (n = 3) showing histology and anti-NEUN (left) or anti-SOX10 (right) staining of 5-μm frozen sections from the corpus callosum (top) and cerebellum (bottom) immediately adjacent to the 50-μm section used for ATAC-seq shown in Supplementary Figure 8. Scale bars, 100 μm. SOX10 staining is shown at a higher resolution due to a more diffuse staining pattern.