Fig. 1: Study overview of Spatial Transcriptomics on murine liver.

a Spatial Transcriptomics was performed on a total of 8 murine liver tissue sections. The tissue sections were placed in one of six, 6.2 × 6.4 mm frames on the glass slide ST array. Each frame contains 1932 spots, with >200 M uniquely barcoded mRNA capture probes. The distance between centers of each neighboring spot is 150 µm (200 µm for spots in the same row). Initially, each tissue section was fixed, stained with hematoxylin and eosin (H&E) and followed by imaging. Then, tissue sections were permeabilized, followed by mRNA capture, tissue removal and sequencing. Thereafter, the count data was subjected to cluster- and differential gene expression analysis (DGEA). The results of the clustering and DGEA were further analyzed and spatially annotated at the global tissue context and down to the lobular level. For new spatial annotations, pathway analysis was performed. Liver lobules are classically described by a central vein (CV, red) surrounded by 6 portal nodes (PV, blue) with neighboring bile-ducts (BD, green). For lobular spatial annotations, clusters have been computationally annotated by comparing expression levels in a set of genetic markers linked to metabolic zonation along the lobular axis. b Canonical correlation analysis (CCA) was performed to integrate data of 8 liver tissue sections, the data was subsequently normalized and subjected to graph-based clustering in which 6 clusters were identified (see Methods). The integrated data was embedded in UMAP space (top) and depicted as an overlay of the spot cluster annotation across the tissue (bottom) (scale bar indicates 500 µm). c Heatmap depicting expression values of the five most variable genes for each cluster after subjecting the 6 clusters to DGEA, with the exception of cluster 3, which resulted in only four significantly differentially expressed genes and cluster 0 which did not result in any significantly differentially expressed genes with the given parameters (Methods). d Visualization of spatial distribution of reported expression markers of Hepatocytes (Alb), liver endothelial cells (Cdh5), Kupffer cells (Clec4f), Cholangiocytes (Spp1), hepatic stellate cells (Reln) and lymphatic liver endothelial cells (Lyve1) by spots under the tissue. Pie-charts indicate the respective proportion of cell type markers present in spots under the tissue (scale bar indicates 500 µm).