Dux4 e55

Myoblasts were grown on 6-well plates as described, washed in PBS, lysed, and scraped in 150 mL RIPA (Pierce/Thermo Scientific Waltham, MA USA, 89900) supplemented with PhosSTOP (Roche Penzberg, Germany, 04 906 837 001) and 1% protease inhibitor cocktail (Sigma P8340). Cells were lysed at 4°C for 20 minutes, centrifuged at 12,000g for 15 min, and the pellet was discarded. Protein concentrations were measured by BIO-RAD DC assay, and 5–10 mg of protein was run. Primary antibodies and dilutions: DUX4 E55 1:1000 (AbCam, Cambridge, UK, ab124699), Vinculin 1:10,000 (Sigma V9131), phosphorylated ribosomal protein S6 1:1000 (Cell Signaling Danvers, MA USA, 4858S), ribosomal protein S6 1:1000 (Cell Signaling 2317S), phosphorylated AKT 308 1:1000 (Cell Signaling 13038P), phosphorylated AKT 473 1:1000 (Cell Signaling 4060S), Pan-AKT 1:1000 (Cell Signaling 4691P), ULK1 1:1000 (Cell Signaling 8054T/S), and LC3A/B 1:1000 (Cell Signaling 12741T). Secondary antibodies and dilutions: anti-rabbit and anti-mouse IgG-HRP 1:2000 (Cell Signaling 7074P2 and 7076S). ECL reagents: Clarity Western ECL Substrate (BIO-RAD, Hercules, CA 102031761) and Clarity MAX Western ECL Substrate (BIO-RAD 76SF121P). Blots were visualized on a BIO-RAD ChemiDoc. Uncropped versions of all western blots are presented in the supplementary materials.

Primary HFF cells were infected with HSV-1 GFP (MOI 1) in PBS supplemented with 0.1% Glucose and 1% FCS. the remaining virus was washed away with a low pH buffer (40mM Citric acid, 10mM KCl, 135mM NaCl, pH3) after one hour at 37°C. CUT&Tag was performed according to the manufacturing protocol (CUT&Tag-IT Assay Kit, Active Motif) using 2,5 μg Dux4 E5–5 (Abcam) or 2,5 μg rabbit IgG (Cell Signalling Technology). In short, cells were bound to concanavalin A-coated magnetic beads and permeabilized for subsequent incubation with primary and secondary antibodies. Specific cutting and addition of adapters was mediated by a protein A-Tn5 transposase fusion protein. Libraries were sequenced on HiSeq 4000 System (Illumina)