Extended Data Fig. 1: Supplemental analysis of the primary model, CRISPRi, and Perturb-seq dataset.

a. (Left) 2D culture of primary human cortical RG showing GFP expression on infection D5. (Right) Immunocytochemical labelling of SOX9, MKI67 and DLX2 before (D0) and after induced differentiation (D3, D7). Representative images from four individuals are shown. b. Barplots showing fractions of SOX2, KI67, EOMES and DLX2 positive populations across the differentiation timepoints (D0, D2, D4, D7) in cells expressing all-in-one CRISPRi vectors with NT sgRNAs. Quantification was done through flow cytometry. Data are presented as mean values +/− SEM. Dots represent independent biological replicates and sgRNAs. D0, n = 2; D2, n = 3 × 2 sgRNAs; D4, n = 3 × 2 sgRNAs; D7, n = 6 × 2 sgRNAs. c. Barplots showing fold changes in fractions of EOMES, ARX and SOX2 positive populations relative to NT in respective KDs to confirm KD efficiency before differentiation (D0), measured by flow cytometry (n = 4 individuals). Dots represent biological replicates from independent individuals and sgRNAs. Two-sided Wilcoxon test on replicate means after collapsing 2 NT sgRNAs; p = 0.00041, 0.00041 (EOMES); 0.00041, 0.0017 (ARX); 0.15, 0.00083 (SOX2). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5X interquartile range; points, outliers. d. Barplot showing fold changes in fractions of SOX2, EOMES and NEUROD2 positive populations measured by flow cytometry in PAX6 and EOMES KD versus NT on D7. Dots represent biological replicates from independent individuals and sgRNAs (n = 4 individuals x 2 sgRNAs). Increase in SOX2 and decrease in EOMES and NEUROD2 populations were detected in both perturbations. Two-sided Wilcoxon test; p values in significant conditions: 0.049, 0.00052, 0.00052, 0.0062 (SOX2); 0.0062, 0.037, 0.00052, 0.00052 (EOMES); 0.00052 (NEUROD2). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5X interquartile range; points, outliers. e. Flow cytometry quantification of fractions of SOX2 positive populations versus NT to confirm efficient KD throughout differentiation (D2, D7). Two-sided Wilcoxon test; p = 0.024, 0.024 (D2); 0.0044, 0.0044 (D7). f. UMAP of cells collected on D0 and D7, coloured by marker gene expression. g. Dotplot of marker gene expression, grouped by differentiation timepoints. h. Violin plot of XIST and RPS4Y1 expression in each individual, highlighting inferred sex genotype. i. Heatmap showing Pearson correlation coefficients of top 25 cell marker gene expression defined in Wang et al.¹² in vivo atlas to datasets from this study (left) and from He et al.⁵⁵ organoid atlas (right). Cell type annotation was done using reference mapping of both datasets to the Wang et al. dataset. j. Heatmap for fraction overlap of labels transferred from reference cell types¹² to the cell types defined in this study. k. Boxplot showing Pearson correlation coefficients in N = 25 (He et al.) and 23 (this study) predicted cell types (diagonal values from (i)). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5X interquartile range. l. UMAP of RNA velocity on D7 NT cells, coloured by supervised cell types. m. Pseudotime tree plots coloured by pseudotime and marker gene expression. n. Violin plots showing gene scores for glycolysis, ER stress, ROS, apoptosis and senescence across six datasets including in vivo human atlas¹², 3D organotypic slice culture, 2D primary RG culture, iNeurons⁵³, primary brain organoids FeBO⁵⁴ and iPSC-derived organoids⁵⁵. Organotypic slice culture data presented at Extended Data Fig. 9 is used here for system benchmarking. o. Histogram showing distributions of singly infected cell counts for each TF-targeting sgRNA (top) and TF (bottom) on D7. p. Dotplot showing target gene KD efficiency compared to NT cells on D7 at the gene level after collapsing active sgRNAs targeting the same gene, colored by significance. log₂FC were calculated with DEseq2 in each cell class in n = 4 individuals. *, p < 0.05; **, p < 0.01; ***, p < 0.001. ER, endoplasmic reticulum; ROS, reactive oxygen species. Scale bars: 200 μm.