A) Analysis of ST data has almost exclusively focused on finding spatial regulation of gene expression, missing potential spatial regulation of RNA processing and splicing. B) An example calculation using Moran’s i for all neighbors (outlined in blue) of the spot outlined in red. The red spot’s score is multiplied with each blue spot’s score individually, and the results are summed and normalized by the number of pairs. The pattern on the left results in a score close to zero, while the pattern on the right results in a score close to one. This indicates that the pattern on the right is more likely to have spots with similar scores grouped together. For the full metric, this calculation is performed over all neighboring pairs (rather than only for the neighbors of one spot). C) To calculate p values, permutations are performed by sampling the scores of each neighboring pair from the scores of that pair in a random gene.

A) For each mouse dataset, Moran’s I for the SpliZ is included on the x axis and Moran’s I for gene expression is included on the y axis. Points with Moran’s I for the SpliZ > 0.2 are labeled. In several genes (marked in blue), Moran’s I for the SpliZ is larger than for gene expression, indicating that the spatial regulation of splicing is more pronounced than the spatial regulation of gene expression. B) Same as A, except Moran’s I for the ReadZS is included on the x axis. C) These plots show only the points from B) corresponding to genomic windows that are significant by the ReadZS but not gene expression. Points for which Moran’s I for ReadZS > 0.15 are labeled.

Splicing spatial regulation plots.

The “histology” column contains histology images of the mouse sections, with anatomy annotated. In the “gene expression” column, each spot is colored according to its quartiled gene expression value. Dark blue corresponds to low gene expression, and yellow corresponds to high gene expression. In the “SpliZ” column each spot is colored by its quartiled SpliZ value. Box plots showing the splicing across each quartile are in the right-most column. For a single splice site (marked with a blue vertical line in the gene annotation), each Visium spot has some fractional usage of the corresponding splice sites (marked by red vertical lines in the gene annotation). Each box plot is created based on these fractions for the Visium spots in the given quartile. The whiskers on the box plots extend to the furthest point 1.5 times the interquartile range in either direction. All other points are marked as outliers. A) Splicing of Myl6 is spatially regulated in the sagittal-posterior mouse brain and the mouse kidney. B) Splicing of Rps24 is spatially regulated in the sagittal-posterior mouse brain and the mouse kidney. C) Splicing of Gng13 is spatially regulated in the sagittal-anterior and sagittal-posterior mouse brain.

3’ UTR usage spatial regulation plots.

The “histology” column contains histology images of the tissue sections, with anatomy annotated where possible. In the “gene expression” column, each spot is colored according to its quartiled gene expression value. Dark blue corresponds to low gene expression, and yellow corresponds to high gene expression. In the “ReadZS” column each spot is colored by its quartiled ReadZS value. Density plots to the right show the read density for the given genomic range, separated by ReadZS quantile. Color bars next to each peak plot indicate which quartile the distribution corresponds to. A) Unannotated 3’ UTRs of Slc8a1 are spatially regulated in the sagittal-anterior and sagittal-posterior mouse brain. Several reads extend past the annotated 3’ end of the transcript. B) Unannotated 3’ UTRs of Gpm61 are spatially regulated in the sagittal-anterior and sagittal-posterior mouse brain. C) Subtle differentiations in the 3’ UTR location of Gpx3 are spatially regulated in the mouse kidney. D) Intron retention of S100A9 is spatially regulated in human tumor brain metastases.

Moran’s I of biological replicates are highly correlated for gene expression, SpliZ, and ReadZS for those genes/genomic windows for which Moran’s I is significant in both replicates.

Biological replicates of Figure 2.

A) Plots of SpliZ vs gene expression for sagittal-anterior mouse brain section 2 and sagittal-posterior mouse brain section 2. B) Plots of ReadZS vs gene expression for sagittal-anterior mouse brain section 2 and sagittal-posterior mouse brain section 2. See Figure 2 for a full description.

Exon expression of Rps8 is spatially regulated in all four mouse brain sections and the mouse kidney. See Figure 4 for a full description.

The 3’ UTR expression of Actb is spatially regulated in all four mouse brain sections and the mouse kidney. See Figure 4 for a full description.