Genetic Manipulation of MUC1, MYC, and NOTCH2

MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma), MYCshRNA (MISSION shRNA TRCN0000039642; Sigma) or a control scrambled shRNA (CshRNA; Sigma) was inserted into the pLKO-tet-puro vector (Plasmid #21915; Addgene). Single guide RNAs targeting MUC1 exon 4 were inserted into the lentiCRISPR v2 (Plasmid #52961; Addgene). Single guide RNAs targeting NOTCH2 were inserted into the lentiCRISPR v2 hygro (Plasmid #98291; Addgene). The viral vectors were produced in 293T cells as described in ref. 19 (link). Cells transduced with the vectors were selected for growth in 1 to 4 μg/mL puromycin or 100 to 400 μg/mL hygromycin. Single cell clones were isolated by the array dilution method. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng/mL doxycycline (DOX; Millipore Sigma).