Computational protocol: Wnt/β-catenin controls follistatin signalling to regulate satellite cell myogenic potential

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[…] Affymetrix MoGene 1.0 ST chipsets (Santa Clara, CA, USA) were RMA normalized [] using the xps in Bioconductor R package [] with the Affymetrix provided MoGene-1_0-st-v1.r4 chip layout and scheme files. The RMA normalized data was log2 transformed and analyzed using the Significance Analysis of Microarrays (SAM) [] method as implemented in the Bioconductor siggenes package []. Heat maps were generated by GenePattern []. Gene ontology of expression data was performed using the functional annotation module of DAVID 6.7 [,]. [...] Total RNA was isolated using the RNeasy Kit and subjected to on-column DNase digestion, as per manufacturer’s instructions (Qiagen, Venlo, Netherlands). cDNA synthesis was performed using the Superscript III reverse transcriptase with random hexamer primers (Invitrogen). SYBR Green quantitative polymerase chain reaction (qPCR) was carried out as previously described []. Transcript levels were normalized to GAPDH transcript levels. Relative fold change in expression was calculated using the ΔΔCT method (CT values <30). PCR primers were designed using the online Primer3 software ( []. Primer sequences are listed in Additional file : Table S1. […]

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