Highlights d In situ sequencing of perturbations or barcodes enables image-based pooled screens d p65 translocation is assayed by imaging in fixed and live cell pools d Pooled live-cell screen identifies MED12 and MED24 as negative regulators of NF-kB

NOTE: This protocol has not been validated with clinical samples. To facilitate collaborations with interested parties to jointly advance the fight against the current coronavirus pandemic, wehave set up a public forum on www.LAMP-Seq.org.

†These authors contributed equally to this work.Large-scale genetic screens play a key role in the systematic discovery of genes underlying cellular phenotypes. Pooling of genetic perturbations greatly increases screening throughput, but has so far been limited to screens of enrichments defined by cell fitness and flow cytometry, or to comparatively lowthroughput single cell gene expression profiles. Although microscopy is a rich source of spatial and temporal information about mammalian cells, high-content imaging screens have been restricted to much less efficient arrayed formats. Here, we introduce an optical method to link perturbations and their phenotypic outcomes at the singlecell level in a pooled setting. Barcoded perturbations are read out by targeted in situ sequencing following image-based phenotyping. We apply this technology to screen a focused set of 952 genes across >3 million cells for involvement in NF-κB activation by imaging the translocation of RelA (p65) to the nucleus, recovering 20 known pathway components and 3 novel candidate positive regulators of IL-1β and TNFα-stimulated immune responses.