Supplementary Figure 4: Direct capture Perturb-seq allows for robust guide assignment and phenotypic analysis in iPSCs with CRISPR cutting (CRISPRn).
From: Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing
a) Box plots displaying the index (guide) UMIs per cell, mRNA UMIs per cell, and transcripts per cell in iPSCs expressing Cas9 (n=5300 cells). Box plots denote quartile ranges (box), median (center mark), and 1.5 × interquartile range (whiskers). b) Median index (guide) UMI counts per cell (capture rate) for cells assigned to each guide identity. c) Heatmap represents gene expression of most differentially expressed genes across all cells with the indicated genetic perturbation, as determined by a random forest classifier. Expression values are the z-scored expression relative to unperturbed cells. Profiles for each gene are calculated by averaging the pseudo-bulk expression profiles of the two independent sgRNAs targeting the gene (n=19 genes targeted by two sgRNAs each). d) Box plot of Pearson correlations of pseudo-bulk expression profiles caused by sgRNAs targeting the same gene (n=19) versus sgRNAs targeting different genes (n=684 pairs). Box plots denote quartile ranges (box), median (center mark), and 1.5 × interquartile range (whiskers). sgRNAs targeting the same gene cause significantly more similar profiles than sgRNAs targeting different genes (Mann-Whitney two-sided U test U=1636.0, p=0.0002). Differences in expression profiles caused by sgRNAs targeting the same genes are likely due to variation of CRISPR cutting efficacy, indel profiles, and/or genetic compensation.
