Figure 4.

 Small RNAs affected by SMCHD1 knockdown in control muscle cells (SMCHD1 signature). (A) SMCHD1 was depleted in control (Control A) myoblasts (MB) and myotubes (MT) using a previously validated SMCHD1 shRNA (19) and a non-silencing control (NSC) shRNA (used as a negative control) in two independent experiments. qRT-PCR analysis of the SMCHD1-depleted cells confirmed SMCHD1 downregulation and induction of the SMCHD1 target genes, PCDHB6 and PCDHB16 shown for a single representative experiment. (B) qRT-PCR analysis of tRNA expression in two independent SMCHD1 knock down experiments (Exp 1 and Exp 2 presented as biological replicates). Three FSHD2-specific tRNAs (tRNA_ValCAC, tRNA_GlyGCC and tRNA_GluCTC) identified in our DESeq2 analysis (Fig. 2H and Supplementary Material, Table S2) were also induced in SMCHD1-depleted control myoblasts, suggesting that differential expression of tRNAs in FSHD2 may be due to SMCHD1 mutations. (C) FSHD2-specific upregulation of the tRNAs tested in (B) was confirmed by qRT-PCR analysis of control and FSHD2 muscle cells. Expression levels normalized to those of GAPDH in the same sample are presented for each experiment as mean ± SD for real-time PCR triplicates.