RNA sequence after electrical stimulation to C2C12 myotubes

We performed electrical pulse stimulation (EPS) in differentiated C2C12 myotubes at low and high frequency, carried out RNA sequencing to address transcriptional regulatory mechanism after EPS. C2C12 myoblasts were seeded into four-well rectangular plates with 3 mL of growth medium comprising DMEM supplemented with 10% fetal bovine serum. Myoblasts culture was maintained in an incubator at 37?C under a 5% CO2 atmosphere. After 2 days, the medium was replaced with a differentiation medium consisting of DMEM supplemented with 2% horse serum. Six days after differentiation, cells were used for EPS. Total RNA was extracted from C2C12 myotubes at 0, 1, 3, and 6 h after 0, 2, or 20 Hz EPS using RNeasy Mini Kit (Qiagen). mRNA was enriched from total RNA using poly(A) selection, and quality of the mRNA samples was validated using 2100 Bioanalyzer (Agilent). Standard Illumina protocols were used to generate 101-base pair, paired-end sequencing reads on the HiSeq 2500 platform (Illumina).