time: day 6 treatment: differentiation treatment: untransduced
Treatment protocol
To generate stable cell lines, MBs were transduced with VSV-G coated shRNA lentiviruses for two days followed by 1 µg/ml puromycin for 3 days. For differentiation of MBs to myotubes (MTs), MBs were plated at 90% confluency in complete media (~3 million cells/8.5 cm plate or 350,000 cells/3.5 cm plate) and permitted to proliferate for 48 h prior to washing in PBS and induction of differentiation by the addition of pre-warmed differentiation media. Differentiation media was changed every 48 h up to 6 days and tetrodotoxin (TTX, Abcam ab120054) was added to a final concentration of 1 µM to prevent muscle contraction 72 h post-serum withdrawl. TTX stocks are generated at 10 mM in 0.1M citrate buffer, pH 4.8 and are stored at -80°C. To specifically isolate differentiated MTs partial trypsinization was performed. Briefly, differentiation media was aspirated and cells subsequently washed once in PBS. TrypLE Express trypsin diluted 1:5 in PBS was then incubated with washed cells for 1 min at 37oC or until MTs, but not mono-nuclear MBs, were released from the growth surface, after which differentiation medium was added to block further trypsin activity and cell detachment. MTs were then carefully extracted by aspirating the cell-containing supernatant after which plates were gently washed in further differentiation medium to gather remaining MTs. To further deplete the sample of MBs, MTs were then preferentially pelleted by limited centrifugation at 500 rpm for 1 min.
Growth protocol
C2C12 Myoblasts (MBs) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin and passaged every 2 days in a 1:7 dilution by trypsinisation. Critically, cells were never permitted to reach more than 80% confluency, i.e. ~2.5 million cells on an 8.5 cm diameter tissue culture plate.
Extracted molecule
total RNA
Extraction protocol
total RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer's instructions
Label
biotin
Label protocol
Ambion Illumina TotalPrep kit
Hybridization protocol
Standard Protocols (Clinical Research Facility Edinburgh)
Scan protocol
Standard Protocols (Clinical Research Facility Edinburgh), Scanner: HiScan H166
Description
replicate 3
Data processing
Data were log2 transformed and quantile normalised using the BioConductor package Limma.
