RNA-seq of the mouse myoblasts cell line C2C12 cultured in four temperature-treatment conditions
RNA-seq 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. Four incubator temperature were set as a treatment condition at 35oC (cold stress), 37oC (control), 39oC (mild heat stress) and 41oC (severe heat stress). The cells were cultured under the treatment conditions for 72 hours prior to harvesting for RNA extraction. The experiment were done in three independent replications.  Cells were lysed in the culture vessel by adding 0.7ml of Qiazol Lysis Reagent, scraped off and further homogenized with syringe and needle according to the manufacturer's recommendation (Qiagen). The RNA was then precipitated with 1 volume of isopropanol, washed with 80% ethanol and finally reconstituted in nuclease-free water. Additional an on-column DNase I treatment was carried out to remove a trace DNA contaminant using RNase-Free DNase Set (Qiagen) and purified using the RNeasy Mini Kit (Qiagen). The RNA quantity and quality was assessed using a NanoDrop ND-1000 (Peqlab) and a Bioanalyzer 2100 (Agilent Technologies), respectively.   2 µg of total RNA with RIN > 8 was used for library preparation using a TruSeq Stranded mRNA Kit with 11 cycles of PCR-amplification according to the manufacture's recommendation (Illumina).