Fig. 5. Registration of spatial data to Allen Common Coordinate Framework and statistical analysis of aligned transcriptomic spots.

A Nonlinear registration of the tissue image from a single brain slice (A₁) and its transcriptomic spot coordinates (A₂)—shown as example: the gene Camk2n1 – to the template image (A₃), slice 70 from the Allen P56 Mouse Common Coordinate Framework (CCF), Allen Mouse Brain Atlas, mouse.brain-map.org. Due to the nonlinear nature of the registration, we were able to precisely align the sample image (A₄) to landmarks in the template image and apply that transformation to the spot coordinates (A₅). To account for different numbers of spots in individual samples, digital spots spaced at 150 µm in a honeycomb were created for the template slice. Each digital spot is populated with the log base 2 normalized transcriptomic counts from the 7 nearest spots from each sample in a group (A₇). This approach allows the comparison of gene expression across entire brain slices in an unrestricted inference space. B–G Samples were split into non-sleep deprived (NSD, n = 6, 42 sample spots per digital spot) and sleep deprived (SD, n = 7, 49 sample spots per digital spot). The range of the color bar for the mean calculations is set from 0 to a log2 fold-change of 3, the maximum fold change for the genes shown, while the color bar for the SD > NSD t-statistic (B3–G3) is bounded to [−4,4], which is approximately the equivalent to the FDR < 0.1. *indicates the gene is significant at FDR < 0.1, **indicates significance at FDR < 0.05. We show a selected group of 6 genes from the 413 DEGs (Supplementary Data 10) (B–G). Panel 1 shows for each gene (B1–G1) the mean normalized gene count in NSD, panel 2 depicts the mean normalized gene count in SD (B2–G2) and panel 3 shows the t-statistics (B3–G3). The following DEGs are depicted: B Per1, 4 significant spots. C Nr4a1, 29 significant spots. D Homer1, 306 significant spots. E Arc, 168 significant spots. F Rbm3, 31 significant spots. G Cirbp, 9 significant spots.