Mab6166

Wells of a 384-well plate were coated with Matrigel (Corning) per the manufacturer’s instructions at 4°C overnight. At time of cell seeding, Matrigel was aspirated, 5 μl of 1% Lipofectamine RNAiMAX transfection reagent (Invitrogen) in Opti-MEM (Gibco) was added per well, and 2.5 μl of 0.5 μM siRNA was added per well, with spin down of plate to ensure settling of reagents. siRNAs were purchased from Dharmacon Inc. as pool of four siRNA targeting sequences per gene. hiPSCs (TL cell line) were seeded as single cells at a seeding density of 20,000 cells/cm², as above, for myogenic differentiation. Final volume per well was 50 μl, including cells, for final siRNA concentration of 25 nM. Twenty-four hours were allowed for cell attachment and transfection before removing plating medium and inducing myogenic differentiation, as above. At day 3, cells were fixed and permeabilized for immunostaining as above and stained for Brachyury (1:100; R&D Systems, AF-2085) and EOMES (1:100; R&D Systems, MAB6166), with secondary and nuclear staining, as above. Fluorescence was imaged with an automated microscope (ImageXpress, Molecular Devices) and quantified with the MetaXpress software (Molecular Devices). There were two biological replicates per experimental condition and four images per well for a total of eight quantified images per experimental condition for average and SD calculations.