8 protocols
AccessionNameType
P-MTAB-26627
bioassay_data_transformation
Data were log2 transformed.
P-MTAB-26626
bioassay_data_transformation
We used loessM normalization. For the loessM normalization we adapted the method of Risso et al. (2009). In order to avoid small values close to 0 the median of the respondent value in the loess estimation is added to the dataset. This modification relaxes the assumption of symmetry among up- and down-regulated genes. We adopted the method for our one dimensional arrays. A brief description and the corresponding R code can be found in http://www.statistik.lmu.de/~kaiser/sup-material.html, respectively. Normalizations were done in R using the function: normalize.loessM (own code, http://www.statistik.lmu.de/~kaiser/sup-material.html).
P-MTAB-26625
image_acquisition
miRNA microarrays were scanned with the Agilent Microarray Scanner G2505C in a single pass mode with a scan resolution of 3 um, 20 bit mode. Signal intensities were extracted and background subtracted using Feature Extraction Software 10.7.3.1 (Agilent Technologies).
P-MTAB-26624
hybridization
Hybridization of labeled miRNAs were carried out by using the miRNA Complete Labeling and Hybridization Kit (Agilent Technologies, Cat. No. 5190-0456) and Mouse miRNA Microarrays Release 15.0, 8x15K (Agilent, G4471A-029152) according to the manufacturer's instructions.
P-MTAB-26623
labeling
Labeling (Cy3) of miRNAs were carried out by using the miRNA Complete Labeling and Hybridization Kit (Agilent Technologies, Cat. No. 5190-0456) according to the manufacturer's instructions.
P-MTAB-26620
grow
Mouse skeletal muscle cells (pmi28) were propagated in Hams F10 (PAA Laboratories), supplemented with 20 % FCS (Sigma-Aldrich), 2 mM L-glutamine (PAA Laboratories), and 1 % Penicillin / Streptomycin (PAA Laboratories). Myoblasts were propagated at 37 C in humidified air (80 % relative humidity) and 5 % CO2. Cells were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium.
P-MTAB-26622
nucleic_acid_extraction
Total RNA was extracted with Trizol (Invitrogen) following manufacturer's instructions.
P-MTAB-26621
specified_biomaterial_action
Cells were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium (DMEM medium containing 2 % horse serum (Gibco), 2 mM L-glutamine (PAA Laboratories), and 0.1 % gentamicin (Gibco)) with 2 x 103 U/ml mouse recombinant TNF-? (Roche Applied Science, Basel, Switzerland), or 5 ug/ml murine recombinant IGF-I, respectively. All media were replenished twice a day. pmi28 cells were harvested 24 h (myoblasts day one and myotubes day one without and with TNF-? or IGF-I, respectively,) after the induction of fusion by serum withdrawal.