RNA FISH and immunofluorescence

For RNA FISH in U-2OS cells, cells expressing the desired RNA (induced for 24 hours) were fixed with 2% paraformaldehyde for 10 min at room temperature and permeabilized by overnight incubation in 70% ethanol at 4°C. Alternatively, cells were fixed and permeabilized by incubation for 10 min in methanol with 10% (v/v) acetic acid. Similar results were obtained with both fixation protocols. Fixed and permeabilized cells were either used immediately, or stored in the permeabilization medium at −20°C until needed. RNA was detected using Cy3 labeled DNA oligonucleotides designed against the MS2-hairpin sequence. Probe sequences are provided in Supplementary Table 2. Hybridization and wash buffers were purchased from Biosearch Technologies and used per the manufacturer’s protocol. For immunofluorescence detection of proteins, methanol fixed cells were stained using antibodies against muscleblind-like-1 (MBNL1, Abcam, ab45899), hnRNP H (Abcam, ab10374), SC-35 (Abcam, ab11826), Coilin (ab87913), Fibrillarin (Abcam, ab5821) and PML (Abcam, ab179466) and a corresponding Alexa Fluor 647 labeled secondary antibody (Invitrogen, A-21236 or Invitrogen A-21244). Samples were co-stained with an anti-GFP booster antibody (GBA488, Bulldog Bio) to visualize RNA foci. After labeling, samples were mounted in Prolong Gold antifade medium (Thermo Scientific, Inc) and imaged using confocal microscopy as described above.