2026-10-15T09:40:22Z https://data.mendeley.com/oai

oai:data.mendeley.com/hhd88xm56z.1 2024-09-10T07:32:25Z

Berry, Scott Poly(A) RNA fluorescence in situ hybridisation (FISH) in HEK293 ARMC5 knockout cells Mendeley Data 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. Fluorescence Microscopy Cellular Imaging Gillis, Alexander Dataset doi:10.17632/hhd88xm56z.1 oai:data.mendeley.com/hhd88xm56z.1 Creative Commons Attribution 4.0 International http://creativecommons.org/licenses/by/4.0 https://data.mendeley.com/datasets/hhd88xm56z 2024-09-10T07:32:25Z