DNA methylation (RRBS) profiling of the mouse myoblasts cell line C2C12 cultured in four temperature-treatment conditions
DNA methylation (RRBS) profiling of the mouse myoblasts cell line C2C12 cultured in four temperature-treatment conditions
The mouse myoblasts cell line C2C12 (ATCC CRL1772) was culture in 75 cm² flask with Dulbecco's Modified Eagle's medium high glucose (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Biochrom) and 1% penicillin-streptomycin as a control condition of 37oC under 5% CO2 until reaching an 80% confluence. The cells were subcultured using 0.125% Trypsin-EDTA (Biochrom) and seeded at 1×105 cells/ well in a 6 well-plate allowed to attach for 30 min in a standard culture condition prior to applying the treatment condition. Incubator temperature was set as a treatment condition at 35°C (cold stress), 37°C (control), 39°C (mild heat stress) and 41°C (severe heat stress). The cells were cultured under the treatment conditions for 72 hours prior to harvesting for DNA extraction. The experiment were done in three independent replications.  Genomic DNA was extracted from samples using a DNeasy kit (Qiagen) according to the manufacturer's recommendation.  2 µg of DNA with a 1% spike-in control (unmethylated cl857 Sam7 Lambda DNA, Promega) was digested for 7 hours with 100U MspI (New England Biolabs) and then overnight with 50U TaqαI. The DNA fragments (double-enzyme digested) were end-repaired, A-tailed and ligated to TruSeq DNA adapters using a TruSeq Nano DNA library prep kit (Illumina) according to the manufacturer's protocols. Size-selection of the adapter-ligated DNA fragments were achieved by using sample purification beads (TruSeq Nano DNA library prep kit) recovering DNA inserts around 40–240bp. The adapter-ligated DNA libraries was subjected to a bisulfite conversion using an EZ DNA Methylation-Gold Kit according to the manufacturer's protocols (Zymo Research). Library amplification was performed using a PfuTurbo Cx Hotstart DNA Polymerase kit (Stratagene). The quality of libraries was assessed on 2100 Bioanalyzer using a high-sensitive DNA chip (Agilent Technologies).