Biospa 8 automated incubator

A RNAi ON-TARGETplus SMARTpool Cherry-pick Library was purchased from Dharmacon/Horizon Discovery, including oligonucleotides targeting all DCAF genes¹⁷ (see Supplementary Table 4 for details). The library was resuspended in 1× siRNA Buffer (Dharmacon, Horizon Discovery) to a final 2 μM stock concentration. HCT-116 and U-2 OS cells, previously transduced with pBabe-mAzG-CCND1, were reverse transfected in a 96-well plate with siRNA:DharmaFECT 2 (Dharmacon, Horizon Discovery) complex using 20 nM siRNA and 0.1 μl of DharmaFECT 2 per well, according to the manufacturer’s instructions. siRNA:DharmaFECT 2 was complexed in Opti-MEM (Thermo Fisher Scientific). Cells were seeded in FluoroBrite DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Corning Life Sciences) and 1% penicillin/streptomycin/l-glutamine (Corning Life Sciences). Images were acquired every 6 h for a total of 72 h using a Cytation 5 Imaging Reader (BioTek), equipped with a humidified BioSpa 8 Automated Incubator (BioTek) set at 37 °C and 5% CO₂. Phase contrast imaging was used to generate a cell mask. Total fluorescence intensity was calculated at each time point within the cell mask, and normalized to the first time point (time = 0). Images were analysed with Gen5 (version 3.0).

Fluorescence polarization assay was performed in 1/2 area black plates (Corning 3686). Reaction mixtures were a total volume of 50 μL and contained final concentrations of 125 nM MSUT2 RNABP and 10 nM FAM-RNA in PBS, transferred using an Integra Viaflo pipet with a 96/50 μL head. Then 2 μL of stock compound was transferred yielding a 10 μM final concentration. Plates were then incubated at room temperature, without shaking, for 20 min in a BioSpa8 automated incubator and transferred by robotic arm to a Biotek Cytation 5 with a preconfigured green polarization filter cube (8040561) at excitation 485/20 and emission 528/20, a dichroic mirror at 510 nm, and a read height of 10 mm. Fluorescence polarization was calculated by first subtracting background from a buffer-only control well and then using the equation $P=\frac{F_{\parallel }-F_{\perp }}{F_{\parallel }+F_{\perp }}$ to determine the polarization (P).