Study design and data analysis.

<p>A) Study design: Next-generation Sequencing (NGS) on three control and three FSHD myoblast cell lines before and after <i>in vitro</i> myogenic differentiation was used in order to derive miRNA modulation in: a) control myogenesis (CN myotubes vs CN myoblasts; arrow a); b) FSHD myogenesis (FSHD myotubes vs FSHD myoblasts; arrow b); c) FSHD myoblasts versus control myoblast (arrow c), and d) FSHD myotubes vs control myotubes (arrow d). B) Flow chart of filtering and analysis of NGS data. NGS generated a total of 153×10⁶ high quality reads, that were filtered for rRNA, tRNA, snRNA, snoRNA, repeat associated RNAs and intron/exon. The filtered reads (approx. 99×10⁶ reads, an average of 8×10⁶/sample) were analyzed to derive known miRNAs (R/Bioconductor) and novel miRNAs (mireap). Differentially expressed miRNAs between samples were derived by log₂FC≧2 and p-value<0.05 parameters. The homogeneity of miRNA modulation among samples was evaluated by cluster analysis (dChip). miRNAs were then validated by qRT-PCR. Finally, target genes were predicted for modulated miRNAs and functionally annotated by DAVID.