Complete skeletal muscle growth medium

Lentiviral particles (MOIs 0.4, 1.6, 6.4, 25.6) were added to the cells culture medium, rocking the plate to ensure homogeneous distribution. Cells were then incubated overnight at 37 °C and 5% CO2. The following day, cells were washed using PBS and cultured for 48 h in complete Skeletal Muscle Growth medium (PromoCell) containing 3 mM GlutaMax (Invitrogen), 10% (v/v) FBS (Life Technology) and 40 μg/ml Gentamicin (Sigma).

Transfection of 2 × 10⁵DUPmyo cells/well of a 6-well plate (10 × 10³ cells/cm²) was performed in serum-free Opti-MEM (Thermo Fisher Scientific) as indicated in manufactures’ instructions. pCMV-GFP plasmid (Addgene #11153) from Connie Cepko’s laboratory was used to test transfection methods. 2.5 μg of plasmid DNA was transfected in combination with different amounts of Lipofectamine 2000 (ThermoFisher Scientific) (6 μl, 9 μl, 12 μl and 15 μl). TurboFect transfection (ThermoFisher Scientific) was done with 2 μg of plasmid DNA mixed with different volumes of transfection reagent (4 μl, 6 μl and 8 μl). 2 μg DNA were also used to transfect cells with 6 μl of GeneJuice reagent (Sigma-Aldrich). After transfection, cells were incubated at 37 °C and 5% CO2 for the amount of time specified by each protocol. Finally, the transfection medium was removed and replaced by the complete Skeletal Muscle Growth medium (PromoCell) and transgene expression evaluated after 48 h.

Transduced/transfected/electroporated cells were washed with PBS and detached from the plate with trypsin–EDTA (Gibco). Cells were centrifuged at 500×g for 5 min at room temperature and washed with PBS. The pellet was resuspended in PBS and transferred into the appropriate Falcon round-bottom polystyrene FACS tubes (Thermo Fisher Scientific). Samples were then placed on ice and brought to the FACS facility (https://www.ucl.ac.uk/child-health/core-scientific-facilities-centres/flow-cytometry- core-facility), where trained personnel performed the FACS by either FACSAria III, FACSCalibur or MoFlow XDP Cell sorter. Sorted cells were then incubated overnight in complete Skeletal Muscle Growth medium (PromoCell), at 37 °C and 5% CO₂.