This study performed loss-of-function screens in a transformed breast cell line using a metabolism-restricted short hairpin RNA (shRNA) library to understand differences in metabolic pathway requirements between in vitro model systems and in vivo tumors. They found that cancer cells depend on high levels of the iron–sulfur cluster biosynthetic enzyme NFS1. This dataset includes supplementary data tied to the publication. The supplementary data contains tables, which includes data for simultaneous in vivo and in vitro screening, curated list of 25 common metabolites utilized by enzymes screened, primary screening for 21 percent oxygen and 3 percent oxygen, and overlap of shRNAs scoring in both screens. The data indicate that lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage.