The one-week automated genome-wide optical pooled screen

Optical pooled screens (OPS) are bottlenecked by labor-intensive in situ sequencing and analysis protocols. Here we present OttoSeq, an automated OPS platform combining the Otto2 fluid handling system with the Brieflow analysis pipeline. We utilized OttoSeq to complete a genome-wide cell painting screen in eight days, sampling more than 5 million high-quality cells across 21,732 gene knockout perturbations (224 cells per gene) and interpreting 320 functional gene clusters.