R elated to Fig. 4 . (A) Filled upper crypt GCs/crypt quantified from AB/PAS-stained tissue sections shown in Fig. 4 D. (B) Percentage of cavitated crypt bases quantified from AB/PAS-stained tissue sections shown in Fig. 4 D. (C) Processing pipeline for determining the expression of age-regulated mucus protein genes (see Fig. 1 H) in GC clusters identified by scRNA-seq. Databases and software used are shown in parentheses. Rn, R. norvegicus; Mm, M. musculus. (D) UMAP plot of scRNA-seq data from isolated colonic GCs highlighting previously annotated GC clusters (Nystrom et al., 2021). (E) Per GC cluster standardized gene expression (z-scores) of secreted proteins (n = 72) identified as significantly enriched in the P9-Adult compared with P1–P7 colonic mucus proteome (see Fig. 1, G and H; and Fig. 4 G). (F) UMAP plot illustrating log₂ expression of Tgm3 in colonic GC scRNA-seq data. Data from n = 5 replicates (A and B) or aggregated from n = 2 independent experiments (D–F). Graphs show median and interquartile range. Statistical comparisons between clusters by two-way (A and B) or one-way (E) ANOVA and Fisher’s LSD; P < 0.0001 (****).