title: GSM2794687: DM3; Homo sapiens; miRNA-Seq
library_construction_protocol: 1 ml of TRIzol was added to each flask. Cells were detached by scraping and homogenized by vortexing and RNA extraction was performed according to the manufacturer's protcol, but using 100% ethanol instead of isopropanol for RNA precipitation (-80°C over night). Total RNA (1 µg) enriched for miRNA was used for generating NGS libraries using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific). Preparation was performed following the instructions for generating small RNA libraries (Ion Total RNA-Seq Kit v2 for Small RNA Libraries, Thermo Fisher Scientific): Adapters were ligated to total RNA molecules followed by reverse transcription. DNA fragments were purified and size selected to enrich fragments of 94 to 114 base pairs. Libraries were post-amplified for 14 cycles and library concentration was determined using the Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific). Equimolar concentrations of up to 16 barcoded miRNA libraries were pooled and sequenced using the Ion Proton System (Thermo Fisher Scientific) with the Ion PI™ Hi-Q™ Sequencing 200 Kit (Thermo Fisher Scientific).