Spatial epigenomic profiling over a whole-mouse brain section. (A) Micrograph displaying the scanning of a mouse brain coronal section deposited on top of a DNA array composed of 64 × 32 interstitial printed probes harboring the mosaic sequence. Three rows of Cy3-labeled probes are visible at the border of the micrograph, corresponding to fiducials used for defining the physical position of the tissue section across the 4096 printed probes. Notice that the DNA array covers a surface of 16 × 8 mm. (B) Micrograph displaying a bright-field scanning of a DNA array printed area in which each of the printed probes is visible as a result of salt deposition. Due to the interstitial printing, the pitch distance resolution of the array is ∼177 microns. (C) Immunolabeling of the section displayed in A, revealing the histone modification H3K4me3. The inset magnification area reveals the nuclear staining visible through this labeling, as well as the differences in cell density across different parts of the mouse brain section. (D) Electropherogram displaying the spatial epigenomic (SpE) Illumina sequencing library obtained from the mouse brain section. (E) Number of total sequenced reads, as well as those recovered at different stages of the primary bioinformatics analysis. (F) Violin plots displaying the number of read counts or promoter regions per spatial epigenomic element (SpExel), in analogy to picture elements (pixels). (G) Digital display corresponding to the number of adjusted read counts associated to the physical positions in the mouse brain section (heatmap displayed in logarithmic scale). (H) Venn diagram displaying the number of promoters presenting H3K4me3 peaks in common between the SpE map and two bulk H3K4me3 ChIP-sequencing profiles (GSM1656749, GSM1000095). For this comparison, all mapped reads across the tissue were collapsed to generate a pseudobulk profile and processed with the peak caller MACS2 (P-value < 1 × 10−5). (I) Peak-centered heatmap for the H3K4me3 peaks retrieved in common between the SpE map and both bulk data sets. Insets display average peak density retrieved in nine peak classes generated from peak clustering based on their shape. (J) Common peaks retrieved between the spatial H3K4me3 profile and the spatial data published by Deng et al. (2022) (GSM5622964). Notice that more than 5000 to 8000 peaks are retrieved in common, on the grounds of the confidence threshold used for the comparison (MACS2 peak calling applied on the corresponding pseudobulk data). (K) Genome browser views assessed around the promoter region associated to the gene adenyl cyclase (Adcy5). (Top) Bulk ChIP-sequencing data from mouse brain samples and visualized within the QC Genomics genome browser. Heatmap on the bottom of each profile corresponds to a local QC enrichment assessment (Mendoza-Parra et al. 2013). (Bottom) Pseudobulk enrichment profiles issued either from the public data set generated by Deng et al. (2022) (GSM5622964; blue) or from the H3K4me3 enrichment map generated in this study (black). (L) Spatial H3K4me3 enrichment signatures retrieved in the promoter region associated to the gene Adcy5. (M) Same as L, but expressed in a differential enrichment context relative to the average levels across the tissue. (N) Mouse brain tissue section analyzed by Deng et al. (2022) and the corresponding H3K4me3 enrichment spatial signature associated to the gene Adcy5. Image adapted from Deng et al. (2022). (O) In situ hybridization (ISH) on a mouse brain section for the gene Adcy5 (Allen Mouse Brain Atlas).
