Fig. 1: A pipeline for deep spatial transcriptomic and proteomic profiling of the mouse hippocampus, and comparative analysis of subregion transcriptomes and proteomes.

a Experimental workflow for tissue analysis. Horizontal brain slices were generated after cutting hemispheres at a 30° angle and subregions or CA1 strata were microdissected and homogenized, followed by parallel RNA-seq and data independent acquisition (DIA) mass spectrometry (see methods). Tissue pooled from 2 mice represented 1 independent biological replicate. SO, Stratum oriens; SP, Stratum pyramidale; SR, Stratum radiatum; SLM, Stratum lacunosum-moleculare. b Schematic indicating subregions CA1, CA2/3 and DG that were microdissected for tissue preparation. c Left: total number of detected mRNA transcripts and quantified protein groups across all subregions (n = 7 replicates). Right: valid value bar plots of quantified protein groups per subregion. Opaque bars indicate proteins quantified in 7/7 replicates. d Principal Component Analysis (PCA) of transcriptome (left) and proteome (right) shows clustering by subregion. Each data point represents one replicate. e Rank abundance plots of mRNA indicating the relative abundance of transcripts in a given subregion. Transcripts with the highest log₂ fold change are ranked as 1. Coloured points represent transcripts significantly enriched in one subregion compared to the others (s value < 0.01, n = 7; see methods for details on normalization and differential expression), while all other detected transcripts are shown in gray. Top 10 transcripts by log₂ fold change in each comparison as well as known markers are labelled. f Gene ontology (GO) overrepresentation analysis based on significantly enriched transcripts from each subregion when compared to both others. Top terms were determined by a one-sided hypergeometric test (p adjusted <0.05). Bubble size corresponds to the number of genes annotated to a given term. g Scatter plots showing log₂ fold enrichment of proteins in each subregion compared to both others. Points are colored by significance (p adjusted <0.01, n = 7; see methods for details on normalization and differential expression) in each comparison. Non-significant proteins detected with 2 or more peptides are shown in grey. h GO overrepresentation analysis based on proteins significantly enriched in each subregion when compared to both others. Top terms and bubble size were determined as in (f). Source data are provided as a source data file.