DMD-targeted RNA-seq data in C25Cl48 muscular cell line treated with siRNA targeting 16 different RBPs

Purpose: The goal of this study was to identify the RBPs that regulate the inclusion of the exons in the muscular isoform of the DMD gene (Dp427m). We compared the splicing pattern of the Dp427m from C25Cl48 treated with a control siRNA or siRNA targeting 16 different RBPs. Method: The C25Cl48 were treated by RNAi in duplicates (or n=4 for the control condition) and submitted to 3 days of myogenic differentiation. The RBPs tested were CELF1, DAZAP1, DDX5/DDX17, hnRNPA1, hnRNPA2, MBNL1, PTBP1, QKI, RBFOX1, RBFOX2, RBM4, SRSF1, SRSF2, TDP-43 and Tra2b. The silencing efficiency was controlled by RT-qPCR and Western blotting analysis. RNA was reverse transcribed using oligodT and the Dp427m (11.3kb) coding sequence was amplified by Long-Range PCR and sequenced on a 454 GS junior platform. Reads that passed quality filters were mapped to the human X chromosome with STAR. The Percent-Spliced-In (PSI) was calculated for each splicing event using intron-centric metrics (Pervouchine et al., 2013), with the SJPIPE pipeline, from the ipsa package. Junctions were filtered to keep only those covered by a minimum of 5 reads in at least one out of the two biological replicates or 2/4 in the control condition. Results: Using this DMD-targeted RNA-seq approach, we obtained an average read depth of 1638X, allowing reliable detection of alternative splicing events. Based on a cutoff of |[delta]PSI|≥0.05, we identified 8 exons that were deregulated in at least one of the RBPs tested, and 15 RNAi conditions induced splicing changes. Conclusion: The 8 deregulated exons identified here are mainly exons that were previously found alternatively spliced in other dystrophin isoforms. si-control and si-RBP treated cells were performed in 4 and 2 biological replicates, respectively.