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. 2018 May 8;27(15):2644–2657. doi: 10.1093/hmg/ddy173

Figure 1.

Figure 1.

 Study design, sample validation and data analysis. (A) Undifferentiated myoblasts (MB) and differentiated myotubes (MT) from two unaffected and two FSHD2 individuals were used to identify small RNAs differentially expressed in FSHD2 during muscle differentiation. (B) Control and FSHD2 muscle cells used in this study were characterized in terms of D4Z4 DNA methylation (Bsa A1/Fse1), D4Z4 repeat size and presence of SMCHD1 mutations. (C) qRT-PCR analysis of the control and FSHD2 MB and MT samples used for small RNA seq showed that the muscle differentiation marker, Actin alpha1, was induced in both control and FSHD2 MT samples and that DUX4 was expressed in FSHD2 samples but not in controls. Error bars in the graph show the SD of the mean for technical triplicates. (D) The flowchart depicting main steps of our small RNA sequencing data analysis. (E) Small RNA libraries were validated by comparing normalized log2 read counts for myogenic and ubiquitous miRNAs between MT and MB samples. Levels of expression of myogenic miRNAs (myomiRs), miR1–1 and miR133A were induced both in control and FSHD2 MT compared to MB, but expression of miR16–1, a ubiquitous miRNA, was not changed during muscle differentiation as expected.