Polysome Profiling of DUX4-Expressing Cells

Polysome profiling was performed as previously described^{63 (link),64 (link)} with the following modifications. Four 70% confluent 15 cm dishes of MB135-iDUX4 cells per condition were treated with 100 μg/mL cycloheximide for 10 min, transferred to wet ice, washed with ice-cold PBS containing 100 μg/mL cycloheximide, and then lysed in 400 μL Lysis Buffer (20 mM HEPES pH 7.4, 15 mM MgCl₂, 200 mM NaCl, 1% Triton X-100, 100 μg/mL cycloheximide, 2 mM DTT, and 100 U/mL SUPERaseIn RNase Inhibitor) per 15 cm dish. The cells and buffer were scraped off the dish and centrifuged at 13,000 rpm for 10 min at 4°C. Lysates were fractionated on a 10%–60% sucrose gradient using the SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter) at 36,000 rpm for 3 h and 10 min. Twenty-four fractions were collected using a Gradient Station ip (BioComp) and an FC 203B Fraction Collector (Gilson) with continuous monitoring of absorbance at 254 nm. RNA from each fraction was extracted using TRIzol LS Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. RT-qPCR was carried out as described above.