Facs aria illu

1.5×10⁵ Caco-2 cells (from ATCC) were plated in 6-well cell culture plates (Corning) and infected with concentrated virus supernatants supplemented with polybrene and additional 500 yp of complete MEM medium. After infecting the cells twice, we selected clones with respective antibiotic selection (Puromycin 4 µg/ml, Sigma-Aldrich; G418-0.8 mg/ml, Merck) from the third day post-infection. We sorted 20% stronger (Fluorescence mean intensity) GFP positive clones with BD FACS Aria Illu (BD Biosciences) with appropriate machine settings, plated them in 6 well plates and expanded. In addition, we assessed the presence of BRAF and KRAS constructs in Caco2-BRAF and Caco2-KRAS cell lines by western blot analysis with rabbit polyclonal anti-GFP antibody at dilution 1:1,000 (A11122, Invitrogen, UK) and BRAFV600E mouse monoclonal at dilution 1:1,000 (VE1, Roche Diagnostics, Indianapolis, IN, USA) (Fig. S1).

Yeast were sorted on a BD FACS Aria Illu, equipped with 405 nm, 440 nm, 488 nm, 561 nm, and 635 nm lasers, and an 85 micron fixed nozzle. Prior to sorting, single-color controls were used to compensate for the minimal FITC overlap with PE. Single cells were gated by FSC vs SSC, and then this population was sorted either by expression (FITC) or by expression and binding (PE). For all sorts, at least ten-fold excess of the library diversity was sorted (∼1.6 million cells for CR9114; ∼500,000 cells for CR6261). For the expression sorts, singlets were sorted into eight equivalent FITC log-spaced gates. For the binding sorts, FITC-positive cells were sorted into 4 PE bins (the PE-negative population comprised bin 1, and the PE-positive population was split into three equivalent log-spaced bins 2–4; see Figure 1—figure supplement 6). Polypropylene collection tubes were coated and filled with 1 mL YPD supplemented with 1% BSA and placed on ice until recovery. Sorted cells were pelleted by spinning at 3000 x g for 10 min, and supernatant was removed by pipette to avoid disturbing the pellets. Pellets were then resuspended in 4 mL SDCAA, a small amount was plated on SDCAA-agar to quantify recovery efficiency, and cultures were rocked at 30°C until reaching late-log phase (OD600 = 0.6–1.2).

Plasmids “pIRES‐hrGFPII”, “IRF2BP1 wild type, siRNA resistant, in pIRES‐hrGFPII”, “IRF2BP1 K579R, siRNA resistant, in pIRES‐hrGFPII” or “IRF2BP1 V578A, siRNA resistant, in pIRES‐hrGFPII” were transfected on 10‐cm plates. After 3 days, cells were split 1:3 and full medium (supplemented with G418) was added. We routinely checked G418 efficiency and used the concentration at which all untransfected HeLa cells died within 5–7 days (varied between 0.5–1.5 mg/ml depending on the age of stock solutions).
After 2 weeks, “low GFP”‐positive cells were sorted using a BD FACSAria Illu™. Usually, only 0.5–5% of the initial cell pools were “low GFP” expressing. FACS sorting was thus repeated several times until the cells appeared stable. We analyzed our polyclonal cell lines once a month by FACS and used them for experiments only if they were at least 70% low GFP expressing.