Extended Data Fig. 7: MERFISH analysis of cell types and Mecp2 expression in Mecp2^KO/+ brain shows expected gene expression and cell distributions across transcriptotypes.

a, Left: transcripts detected (colored dots) and DAPI images of Mecp2^KO/+ MERFISH data in one experiment. Scale bar is 250 μm for low resolution view, and 25 μm for close-ups. Middle: correlation of mean log2 CPM (across all detected cells) of each gene for experiments 1 and 2. Right: correlation between each pair of MERFISH experiments in log2 mean CPM of each gene. b, UMAP of MERFISH cells, colored by experiment identity and biological replicate. c, Log-transformed CPMs of expression for each gene probed by MERFISH in neuronal subclasses compared to its expression detected by INTACT RNA-seq. d, Type and subclass numbers in MERFISH experiments. e. Z-scores of average expression for each gene in the MERFISH panel in each cell type. Inset: close-ups of Pvalb (PV marker gene), Fezf2 (L5 PT CTX marker gene), and Rorb (L4/5 IT CTX marker gene). f, Representative distributions of transcript counts per cell for Mecp2 and a representative negative control (‘Blank counts’) detected in PV cells in MERFISH analysis of wild-type or Mecp2^KO/+ coronal sections. Gray = thresholds used for calling ‘WT’ and ‘KO’ transcriptotypes. g, UMAP of MERFISH cells colored by transcriptotype. h, MERFISH log2 fold-change in expression of genes identified as MeCP2-repressed in PV, SST, L4, and L5 INTACT RNA-seq analyses. ****p < 0.0001 two-sided Wilcoxon rank-sum test. i, WT, Mecp2 KO, or unassigned cell proportions of a subclass or all cells, for each biological replicate. ns=not significant, *p < 0.05, **p < 0.01 two-sided paired t-test. j, Locations of WT and KO cells of subclasses across cortical layers in the visual cortex identified using MERFISH. k, Comparing the WT fold-differences in expression of type-specific DEGs between closely related types of the same subclass to fold-differences between Mecp2 KO and WT cells of MeCP2-repressed genes or non-DEGs (as in Fig. 5h). Comparison of the magnitudes of differences shows that loss of MeCP2 changes leads to only a partial loss of differences in type-specific DEGs between closely related types. High/low: log2 fold-difference in expression of previously defined type-specific DEGs² between types within a subclass. KO/WT: log2 fold-difference between Mecp2 KO and WT of MeCP2-repressed genes lowly expressed (‘Low MR’) or highly expressed (‘High MR’) in types relative to related types, or non-DEGs. MERFISH data are from n = 3 bioreplicates across 4 imaged brain sections from 8–10-week-old female Mecp2^KO/+ mice. Boxplots represent data as described in Fig. 2. ρ = Spearman’s correlation coefficient. B1-3=biological replicate 1-3.