The supplemental information provides detailed experimental validation and analysis of circular RNA (circRNA) ZNF609 in myogenesis. Key points include: 1. **RNAseq Analysis**: Figure S1 shows RNAseq data from human and mouse myoblasts and myotubes, highlighting gene expression changes and correlation between replicates. Gene ontology enrichment analysis and qRT-PCR validation of myogenic markers are also presented. 2. **Circularized Exons**: The study identifies circ-CDYL, circ-QKI, and circ-ZNF609 as circularized exons. Rolling circle RT-PCR and melting curve analysis validate these circRNAs. 3. **qRT-PCR Validation**: qRT-PCR is used to validate the expression of selected circRNAs in human and mouse myoblasts and myotubes. 4. **Oligo dT Purification**: Oligo dT purification of total RNA is compared with random priming, showing the recovery of circular and linear isoforms. 5. **Subcellular Localization**: RT-PCR is used to determine the subcellular localization of selected circRNAs, confirming their presence in both nuclear and cytoplasmic fractions. 6. **Knock-down Screening**: A high-throughput screening experiment is conducted to knock down circRNAs and linear mRNAs, with validation of the knock-down efficiency and phenotypic effects. 7. **Circ-ZNF609 Phenotype Validation**: The phenotype of circ-ZNF609 knock-down in mouse cells is validated, showing reduced BrdU incorporation and altered gene expression. 8. **Association with Polysomes**: Circ-ZNF609 is shown to associate with polysomes, indicating its potential for translation. 9. **Translation of Circ-ZNF609**: Western blot and immunofluorescence analyses demonstrate the production of proteins by circ-ZNF609, and Northern blot and RT-PCR confirm the presence of the tagged circRNA in neural differentiated cells. 10. **m6A CLIP Sites**: m6A CLIP sites in circ-ZNF609 are identified and validated, showing their presence in C2C12 myoblasts. The supplemental information supports the functional role of circ-ZNF609 in myogenesis and its potential for translation and regulation.The supplemental information provides detailed experimental validation and analysis of circular RNA (circRNA) ZNF609 in myogenesis. Key points include: 1. **RNAseq Analysis**: Figure S1 shows RNAseq data from human and mouse myoblasts and myotubes, highlighting gene expression changes and correlation between replicates. Gene ontology enrichment analysis and qRT-PCR validation of myogenic markers are also presented. 2. **Circularized Exons**: The study identifies circ-CDYL, circ-QKI, and circ-ZNF609 as circularized exons. Rolling circle RT-PCR and melting curve analysis validate these circRNAs. 3. **qRT-PCR Validation**: qRT-PCR is used to validate the expression of selected circRNAs in human and mouse myoblasts and myotubes. 4. **Oligo dT Purification**: Oligo dT purification of total RNA is compared with random priming, showing the recovery of circular and linear isoforms. 5. **Subcellular Localization**: RT-PCR is used to determine the subcellular localization of selected circRNAs, confirming their presence in both nuclear and cytoplasmic fractions. 6. **Knock-down Screening**: A high-throughput screening experiment is conducted to knock down circRNAs and linear mRNAs, with validation of the knock-down efficiency and phenotypic effects. 7. **Circ-ZNF609 Phenotype Validation**: The phenotype of circ-ZNF609 knock-down in mouse cells is validated, showing reduced BrdU incorporation and altered gene expression. 8. **Association with Polysomes**: Circ-ZNF609 is shown to associate with polysomes, indicating its potential for translation. 9. **Translation of Circ-ZNF609**: Western blot and immunofluorescence analyses demonstrate the production of proteins by circ-ZNF609, and Northern blot and RT-PCR confirm the presence of the tagged circRNA in neural differentiated cells. 10. **m6A CLIP Sites**: m6A CLIP sites in circ-ZNF609 are identified and validated, showing their presence in C2C12 myoblasts. The supplemental information supports the functional role of circ-ZNF609 in myogenesis and its potential for translation and regulation.