Extended Data Fig. 1: Genome-wide screen quality control.
From: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells
a, b, Normalized count matrices of all sgRNA counts across samples at Day 4 (a) or Day 14 (b). c, Growth curves with number of cells at each passage (p3-p12) for primary cultures of NSCs from young (3-4 months) and old (18–21 months) mice used for the genome-wide screens. Dots represent cell counts for the sample at each passage (x106). Dotted line at 1.4×109 represents the required number of cells for each biological replicate. d-f, Pairwise comparison of CasTLE scores across young screen samples at Day 14. Correlation plots between Screen 1 and Screen 2, Screen 2 and Screen 3; Screen 1 and Screen 3. Spearman ρ is indicated. g-i, Principal Component Analysis (PCA) performed on all gene scores of the three independent screens at Day 4 (g,h) and Day 14 (i): Young 1, 2, 3 (blue) and Old 1, 2, 3 (red), with Principal Components 1 vs. 2 (g) and Principal Components 3 vs. 4 (h,i). j,k, Volcano plots of example screen results (screen 1) at Day 14 for young (j) or old NSCs (k), showing gene scores as a function of effect size. Each dot represents one gene. Labelled dots are top ranking gene knockouts in at least 2 of the 3 independent screens (FDR < 0.1). Selecting genes that intersect screen 1 (day 4 or 14) with screen 2 (day 4 or 14) that boost NSC activation (purple, corresponding to enriched sgRNAs) or impede NSC activation (green, corresponding to depleted sgRNAs) in an age-dependent manner or gene knockouts that boost activation regardless of age (grey, corresponding to enriched sgRNAs). See Supplementary Table 1 for complete list of gene scores. l, Comparison of significantly depleted genes (FDR < 0.1) in genome-wide screen (Day 14) and essential genes identified from Online GEne Essentiality database (OGEE)92. m, Comparison of significantly depleted genes (FDR < 0.1) in genome-wide screen (Day 14) and essential genes identified from Core Essential Genes 2 (CEG2)91. n, Validation of gene knockout efficiency at the genomic level. qNSCs were infected with lentivirus expressing sgRNAs targeting individual genes (5 sgRNAs per gene) and genomic DNA was extracted. Percentage of knockout was quantified by sequencing PCR products followed by DECODRv3.0. Top: Experiment 1. Dot plot of the percentage of knockout for n = 2 independent primary NSC cultures, each derived from 2 mice (one young, one old). Connecting line indicates range. Bottom: Experiment 2. Dot plot of the percentage of knockout for n = 1 independent primary NSC culture derived from 1 mouse (young). Each dot represents the percentage knockout for one sgRNA. #: knockout detected by DECODRv3.0, but with low confidence (r2 < 0.6) (see Source Data). No data point: knockout not detected by DECODRv3.0 (see Source Data). Dotted red line: sum of knockout percentages for high confidence and detected knockouts. See Extended Data Fig. 6h–m for genomic knockout examples and knockout efficiency by western blot and FACS for Slc2a4 (GLUT4).
