5-Ethynyl Uridine nascent RNA labelling in HCT116 cells (ARMC5 knockdown)

Description

siRNA transfections. In 96-well plates, 25 µL of siRNA at 30 nM in Opti-MEM (Thermo Fisher 31985-062) was added per well, followed by 25 µL of transfection reagent (Thermo Fisher Lipofectamine RNAiMAX 13778100) diluted 1/125 in OptiMEM. In 384-well plates, 10 µL of siRNA at 30 nM in Opti-MEM was added per well, followed by 10 µL of diluted transfection reagent. After 20-30 minutes of room temperature incubation, HCT116 cells were added onto the transfection reaction and allowed to settle. Experiments were conducted three days after siRNA transfection. Assays were performed as previously described (https://doi.org/10.1016/j.cels.2022.04.005). All steps were followed by three PBS washes. Cells were fixed in 4% paraformaldehyde (EMS Emgrid 15710) for 15 minutes, then permeabilised in 0.25% Triton X100 (Sigma Aldrich 93443) for 10 minutes. Cells were changed into Tris-buffered saline (125 mM sodium chloride, 50 mM Tris pH 8 Thermo Fisher Invitrogen AM9856) via 3x wash. A 1.5x click reaction mixture was made in TBS containing 150 mM sodium ascorbate (Sigma Aldrich A7631), 3 mM copper sulphate (Chem-Supply Australia CA068) and 7.5 µM Alexa647 azide (Thermo Fisher Invitrogen A10277). 30 µL of 1.5x click reaction was added onto 15 µL residual TBS and incubated for 30 minutes at room temperature. Total protein was stained with 1 µM Alexa488-NHS in 50 mM carbonate buffer at a pH of 9.2 for 15 minutes, with DAPI added for 5 minutes at 200 ng/mL in PBS. Imaging was performed on a Nikon Ti2 microscope equipped with a Yokogawa CSU-W1 spinning disk, with 40x/NA0.95 Plan Apo λ air objective, and dual Hamamatsu ORCA-Fusion C14440-20UP cameras. 20 z-planes at 1 µm intervals were acquired. DAPI DNA stain was acquired with a 405 nm laser and 450/82 nm filter. Alexa488+ conjugated secondary antibodies were acquired with a 488 nm laser and 525/50 nm filter. Alexa647 (EU) was acquired with a 647nm laser and 685/40 nm filter.

Steps to reproduce

- Folders 20240701_175159_151, etc. contain single-cell quantifications over time (QUANTIFICATION subfolder) and imaging metadata (OME-TIFF-MIP subfolder) - hct116_EU_analysis.html shows all data analysis steps from the single-cell data provided and generates PLOTS and SUMMARIES - hct116_EU_IF_analysis.html shows all data analysis steps for RPB1 immunofluorescence performed on the same samples as EU experiments.

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