Sample preparation kits

Primary myoblasts stably expressing empty vector, FLAG-PAX3, or FLAG-PAX3-FOXO1 were grown to approximately 80% confluency. Total RNA was isolated using the miRNeasy mini kit (Qiagen), allowing for the isolation of RNA <30 bp in length, according to the manufacturer's specifications. Poly-A⁺ mRNA or miRNA were isolated from 4μg total RNA, to generate the respective cDNA libraries, both using the Illumina sample preparation kits according to the manufacturer's specifications. The cDNA libraries were provided a unique index identifier, allowing the clustering of several samples into a single sequencing lane, and deep sequencing analyses were performed in triplicate from three independent cell growth, RNA isolation, and cDNA library constructions.

The DGE libraries for five samples were processed in parallel using Illumina sample preparation kits. Briefly, mRNA was captured from total RNA of each sample with magnetic oligo (dT) beads. Following first and second strand cDNA synthesis, Endonuclease NlaIII was used to digest the bead-bound cDNA, and bound fragments containing a CATG sequence site adjacent to the poly (A) tail at the 3’ end were acquired. After precipitation of the 3’ cDNA fragment, Illumina adaptor 1 was added to the 5’ end; this adaptor contains a recognition site for the endonuclease MmeI to cut 17 bp downstream of the recognition site (CTAG) and produce 17 bp tags with adaptor 1. Illumina adapter 2 was introduced at the site of MmeI cleavage after removing the 3’ fragment via magnetic bead precipitation. The tags with both adapter 1 and adapter 2 were then prepared for Illumina DNA sequencing [35 (link)].

Genomic DNA was extracted as described (Rancati et al., 2008 (link)). Sequencing libraries were prepared using Illumina sample preparation kits and indexed paired-end (PE) sequences were obtained on an Illumina HiSeq platform. All sequencing data were deposited in NCBI SRA database under accession number SRP056269. Data pre-processing, mapping and variant calling was performed in CLC Genomics Workbench, essentially as described (Liu et al., 2015 (link)). Higher level data integration and analysis was performed via customized R scripts. Detailed sequencing protocols and analysis parameters are provided in Supplemental Methods. A summary of the sequencing data and results is reported in Supplementary Tables 3, 4.