Extended Data Fig. 4: Multimodal PerturbView screen in primary immune cells.
From: Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView
a, PerturbView enables efficient sgRNA recovery after different multiplexed imaging techniques. Left, BMDMs were stained with F4/80 repeatedly using 4i, IBEX and cycIF (Scale Bar, 50 μm). Middle, Representative images of in situ sequencing results after 6 rounds of staining (Scale Bar, 50 μm). Right, Mean percentage of cells positive for sgRNA signal (y axis, ≥1 sgRNA read per cell, mean of well replicates) after each staining step (x axis). Error bar, SD. n = 2 independent replicates. b, Agreement in phenotypic profiles of different guides targeting the same gene. PHATE embeddings of RNA/protein joint profiles (dots) for each sgRNAs in the TNFα (left) or IL1β (right) screen targeting a gene (color) or controls (grey). Gene names are shown for each guide whose 5 nearest neighbors contain another guide targeting the same gene. c, Distribution of cosine similarity of the phenotypic profiles derived from joint RNA/protein profiling for random pairs of guides (“random”, blue) and either guides targeting the same gene across all genes (orange; “same gene”, top) or targeting Olfr genes (orange “same gene”, bottom). d, e, Contribution of different molecular phenotypes to perturbation impact. d, Impact score (FDR based on one-sided tests adjusted with Benjamini-Hochberg method, color bar) for each perturbed gene (row) in the TNFα (top) or IL1β (bottom) screen, when assessed only based on one imaging feature (single-channel, left) or all other features (dropping a channel, right). Rows and columns are clustered by hierarchical clustering. e, Top, CDFs of phospho-rpS6 intensity (x axis) in response to TNFα for four guides (colored curves), compared to cells with non-targeting or non-essential controls (gray line (mean); shading standard deviation). FDRs were computed for the genes listed (in the rows) in d. Bottom, representative phospho-rpS6 images under perturbations. Two cells were sampled from each of a 10-percentile group (according to the mean cellular phospho-rpS6 level) and arranged from low (left) to high (right).
