CROPseq-iT7 backbone and Brunello library were cloned, tissue culture conditions for various cell lines were established, and library transduction and quality control were performed. NIS-Seq perturbation screens were conducted, and imaging and image analysis were detailed. Single-gene perturbation cell lines were generated for validation, and primary human macrophages were differentiated, transduced, and nucleofected. Inflammasome stimulation and human epidermal sheet preparation, transduction, and sequencing were also described. Zero-knowledge image analysis using deep learning was performed, and ethics statements were included.