Illumina HiSeq 4000 sequencing; miRNA expression profile in response to SDF-1 treatment
miRNA expression profile in response to SDF-1 treatment
SC-derived myoblasts were transfected with predesigned Silencer Select siRNA (Life Technologies) complementary to CXCR4 encoding mRNA (ID: s64091). Appropriate negative control siRNA was used, according to the manufacturer's recommendation. The siRNA duplexes were diluted in DMEM to a concentration of 100 pmol and Lipofectamine RNAiMAX (Life Technologies) was added according to the manufacturer's instructions. Mouse recombinant SDF-1 (10 ng/Âµl) was added to cell culture 24 h after transfection. The cells were then collected and processed 48h after SDF-1 treatment for RNA isolation and RNA sequencing. RNA was extracted in AROS Applied Biotechnology A/S Science Park Skejby (Denmark) with the QIAsymphony RNA extraction kit from QIAGEN (Germany) using the QIAsymphony SP Biorobot from QIAGEN (miRNA CT 400 program). MiRNA was quantified using a Qubit fluorometer and approximately 150 bp-long barcode libraries were created. The quality of the libraries was validated using the Bioanalyzer 2100 DNA High Sensitivity Kit (Agilent, USA). Libraries' concentrations were measured with the KAPA Library Quantification Kit during qPCR. Libraries were pooled with a 30% increase in control libraries PhiX (Illumina) and hybridized to a sequencing flow cell.