Neon transfection system starter pack

CRISPR Cas9-mediated MARCKS knockout was generated by Cas9 ribonucleoprotein using the Neon Transfection System Starter Pack (Invitrogen) according to the manufacturer’s instruction. Briefly, three single guide RNA (sgRNAs) that contain MARCKS targeting sequences and a Cas9 nuclease-recruiting enzyme were designed using CRISPR-Cas9 guide RNA design checker (www.idtdna.com/CRISPR-Cas9). IMMs were collected and washed once with PBS. Then, 4×10⁶ cells were electroporate with Cas9 protein v.3 (Integrated DNA technologies, San Diego, CA, USA), complexed with 3 sgRNAs (seed sequences: 5’-CACGTCGTCGCCCAAGGCGG-3, 5’-TGGCCACGTAAAAGTGAACG-3’, 5’-AGCAAGAAGGAGTCGGGCGA-3’) in the nucleofector buffer (Invitrogen, Waltham, MA, USA) and cultured for 3–4 days. The knockout candidates were screened using western blot and mass spectrometry.

MyoG promoter (+3 ~ −1596), MyoG promoter + E1 (WT or Mutation), and MyoG promoter + E2 (WT or Mutation) were cloned into pGL3 basic vector and transfected to primary myoblasts using the Neon™ Transfection System Starter Pack (Invitrogen, MPK5000S), respectively. After transfection 48 h, luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega, PR-E1910) by BioTek Synergy NEO (BioTek) following the manufacturer’s instructions. Relative luciferase activity was calculated as the ratio of Firefly/Renilla luciferase activity. All experiments were repeated at least 3 times. Primers were listed in Tables S3 and S4.