Cell Culturing Techniques and Cell Lines

All cells were grown in an atmosphere of 5% carbon dioxide (CO₂) at 37 °C. Unless indicated otherwise base media were supplemented with 10% heat-inactivated fetal bovine serum (Serum Plus II; Sigma-Aldrich) and 1% penicillin/streptomycin and L-Glutamine (Mediatech Inc.). Cells were dissociated with 0.25% (w/v) Trypsin—0.53 mM EDTA solution (Mediatech Inc.). 293T parental and Dicer knock out cells (clone 4–25, provided by Dr. Bryan Cullen, Duke University) (RRID:CVCL_0063) were cultured in DMEM (Cellgro). The HCT116 Ago 1/2/3 k.o. cells [53 (link)] were provided by David Corey (UT Southwestern). HCT116 Dicer k.o. cells were purchased from the Korean Collection for Type Cultures (KCTC, clone #43, cat #HC19023) and cultured in McCoy 5 A medium. Neuroblastoma cell line NB7 [54 (link)] was cultured in RPMI1640. Ago2 ko 293T cells (provided by Dr. Klaas Mulder, Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands) and HeLa wt and Ago2 ko cells [55 (link)] (provided by Dr. Sarah Gallois-Montbrun, Université Paris Descartes, Paris, France), were all cultured in DMEM (Cellgro). Lipofectamine RNAiMAX was from ThermoFisher Scientific (#13778150).