Poly(A) RNA fluorescence in situ hybridisation (FISH) in HEK293 ARMC5 knockout cells

Description

Assays were performed similarly to previous descriptions (https://doi.org/10.1016/j.cels.2022.04.005). All steps were followed by three PBS washes (cell fixation and permeabilisation) or 2X saline sodium citrate (SSC) buffer (FISH steps; Thermo Fisher Invitrogen AM9763). Cells were fixed in 4% paraformaldehyde (EMS Emgrid 15710) for 15 minutes, then permeabilised in 70% ethanol at 4 ºC for 4-6 hours. Total protein was stained with 1 µM Alexa488-NHS in a 50 mM carbonate buffer at a pH of 9.2 for 15 minutes. Cells were washed twice with FISH wash buffer containing 10% formamide (Thermo Fisher Invitrogen AM9342) in 2X SSC. Cells were hybridised overnight at 37 ºC with 100 nM ATTO647N-labelled poly-dT (Integrated DNA Technologies) in a hybridisation buffer containing 10% formamide by volume (Thermo Fisher Invitrogen AM9342), 2 mM ribonucleoside vanadyl complexes (New England Biolabs S1402S), 100 µg/mL yeast transfer RNAs (Thermo Fisher Invitrogen 15401011), 200 µg/mL BSA (Thermo Fisher Invitrogen AM2616), and 100 mg/mL dextran sulphate (Merck Sigma Aldrich D8906-50G) in 2X SSC. The next day, two one-hour washes at 37 ºC were performed in FISH wash buffer, the second containing DAPI at 200 ng/mL. A single room temperature wash in FISH wash buffer was performed, followed by washing three times in 2X SSC alone, which cells were left in for imaging.

Steps to reproduce

- Folders 20240315_142116_487, etc. contain single-cell quantifications over time (QUANTIFICATION subfolder) and imaging metadata (OME-TIFF-MIP subfolder) - hek_polyA_analysis.html shows all data analysis steps from the single-cell data provided and generates PLOTS and SUMMARIES

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