*2.2. Derivation of C2-YAl-KO Clones*

To delete the exon 3 of NF-YA gene in C2C12 cells, four guide RNAs (gRNAs) were designed to simultaneously target the two flanking introns by using the online tool https://zlab.bio/guide-designresources. Potential off-target sites were monitored by the online tool https://crispr.cos.uni-heidelberg. de: Table S1 shows the results of such analysis for the four guides. The selected gRNAs had no common off-target sites and were cloned in the two plasmids pX330A\_D10A-1x2\_ac and pX330A\_ D10A-1x2\_bd, following the Multiplex CRISPR/Cas9n Assembly System Kit protocol [42]. 1 <sup>×</sup> 106 C2C12 cells were transfected with 3 μg of the two gRNAs/CRISPR/Cas9n plasmids by electroporation and plated at low density. 72 h after transfection, single clones were picked, expanded and screened.

For DNA extraction, cells from the individual clones were washed with PBS, collected by scraping, lysed in 100 μL ice-cold lysis buffer (40 mM Tris-HCl, 2 mM EDTA, 0.08% SDS, 80 mM NaCl, 0.5 μg/μL Proteinase K) and incubated overnight at 37 ◦C in agitation. To precipitate DNA, 100 μL of ice-cold 2-propanol and 0.3 M NaAc were added, samples were mixed and centrifuged at 13,000 rpm for 30 min at 4 ◦C. The pellet was washed with 150 μL of 70% ethanol, centrifuged at 13,000 rpm for 30 min at

4 ◦C. Supernatant was discarded, the pellet was dried and resuspended in 30 μL H2O. The resulting DNAs were then screened for positive exon 3 deletion by PCR.

We screened a total of 335 individual clones and obtained 2 independent homozygously edited clones.

#### *2.3. Protein Extraction and Western Blot Analysis*

For Whole Cell Extracts preparation, cells were pelleted by centrifugation, resuspended in ice-cold RIPA buffer (10 mM TrisHCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.1% sodium deoxycholate, 140 mM NaCl, 1% Triton X-100, 1 mM PMSF, Protease inhibitor cocktail) and incubated for 30 min on ice, with occasional shaking. Samples were centrifuged at 13,000 rpm for 10 min at 4◦C and the supernatant recovered and quantified using the Bradford protein assay.

20 μg of extracts were loaded on a 4–10% SDS-polyacrylamide gel and analyzed by Western blot using primary antibodies and a peroxidase-conjugate secondary antibody (Sigma-Aldrich). Primary antibodies: anti-NF-YA (G-2, Santa Cruz), anti-NF-YB (GeneSpin), anti-NF-YC (home-made) anti-Vinculin (H-10, Santa Cruz), anti-MyHCs (MF20, DHSB), anti-Myogenin (IF5D, DHSB), anti-MyoD (C-20, Santa Cruz), anti-Myf5 (C-20, Santa Cruz), anti-Pax3 (DHSB), anti-Snai1 (C15D3, Cell Signaling). Western blot experiments were performed on three independent biological replicates.

#### *2.4. Reverse Transcriptase PCR and Real-Time PCR*

RNA was isolated by the Tri Reagent (Sigma-Aldrich) protocol according to the manufacturer's instruction. The cDNA was produced starting from 1 μg of total RNA using the MMLV Reverse Transcription Mix (GeneSpin) and used for real-time PCR (SYBR® Green Master Mix, Bio-rad Laboratories) analysis. Real-time PCRs were performed with oligonucleotides designed to amplify 100–200 bp fragments (Table S2). The housekeeping gene Rsp15a was used to normalize expression data. The relative sample enrichment was calculated with the formula 2–(ΔΔCt), where ΔΔCt = [(Ct sample – Ct Rps15a)x − (Ct sample – Ct Rps15a)y], x = sample and y = sample control. RT-qPCR analyses were performed on three independent biological replicates. For ChIP experiments, we figured out the percentage of input immunoprecipitated by NF-YB and nc (negative control) antibodies. Results of three independent experiments were represented as Fold change (Fc) between NF-YB sample and nc sample as: %Input NF-YB/%Input nc.

#### *2.5. Flow Cytometry Analyses*

Cells were harvested by trypsinization and washed in PBS, fixed in ice-cold 70% ethanol and stored at 4 ◦C at least 24 hours. Cells were then washed with 1% BSA in PBS and resuspended in 500 μL of PI-staining solution (20 μg/mL Propidium Iodide, 10 μg/mL RNaseA, 1X PBS) at room temperature for 30 minutes, light protected. FACS analyses were performed using the BD FACSCantoII, analyzed with FACSDiva software and quantified with FlowJo. A total of 10<sup>4</sup> events were acquired for each sample. Three independent FACS experiments were performed.

#### *2.6. Immunofluorescence*

For immunofluorescence analyses, cells were washed three times with PBS and fixed 10 min with ice-cold acetone-methanol (1:1) at room temperature. After three washes, cells were permeabilized with 0.25% Triton X-100 in PBS for 5 min and incubated 1 h with the primary antibody anti-sarcomeric MyHCs (MF20, DHSB) at room temperature. Cells were washed three times, permeabilized 5 min with 0.25% Triton X-100 in PBS and incubated with secondary FITC anti-mouse antibody (1:500, Sigma-Aldrich) plus DAPI (2 μg/mL) for 40 min at room temperature, light protected. The acquisition was performed by using the inverted microscope Leica DMI6000 B. Three independent immunofluorescence experiments were performed.

#### *2.7. Overexpression and RNA Interference Experiments*

Myogenin overexpression was performed by electroporating 1 <sup>×</sup> 106 C2C12 cells with 3 <sup>μ</sup>g of plasmid (pEMSV-Empty/pEMSV-Myog) and plating them in DM for 96 h. Cells were then collected and analyzed. Three independent biological replicates were performed.

For small interfering RNA (siRNA)-mediated knockdown of NF-YB [29], 2 <sup>×</sup> 106 C2C12 cells were transfected by electroporation with 100 nM of NF-YB [29] or scrambled control siRNA (Qiagen, SI01327193) and plated into a 10 cm plate in GM condition. 72 h after transfection, cells were collected by scraping for total protein preparation and RNA extraction. Gene expression was analyzed performing real-time PCR. Two independent biological replicates of siRNA interference were performed.
