Quantifying DUX4 and LEUTX mRNA in FSHD2 Myoblasts

FSHD2-2 myoblasts were seeded in micro-slide eight-well plates at ~8x10⁴ cells per well, and differentiation was initiated ~20hrs later. After 3 or 7 days, as indicated, of differentiation, cells were fixed with 10% neutral buffered formalin at room temperature for 30 min, and the RNA FISH experiments were performed using the RNAScope fluorescent Multiplex system (Advanced Cell Diagnostic Inc.) according to the manufacturer’s instructions. For costaining of immunofluorescent (IF) staining and RNAScope, cells were permeabilized with 0.5% Triton X-100 for 5 min at 4°C between fixation and dehydration process, then DUX4 (Abcam, ab124699) IF was performed as previously described [40 (link)]. Probe-Hs-DUX4-C1, Probe-Hs-LEUTX-C2, were custom-designed to avoid crossreactivity to related homologs (for DUX4 probe set, see S11A Fig). Probe-Hs-SLC34A2-C3 was also used. Images were acquired using a Zeiss LSM 510 META confocal microscope. A technical consideration should be made that due to the process of IF and RNAScope costaining that much of the cytoplasmic RNAScope signal is washed out.