title: GSM4565666: Samples-1-61-AGGCAATG; Homo sapiens; RNA-Seq
library_construction_protocol: Approximately 10 mg of skeletal muscle tissues were homogenized in a Bead Ruptor Elite bead mill homogenizer (Omni International, Kennesaw, GA) cooled by liquid nitrogen and homogenized for 2 x 20 seconds at 4.2 m/s. Muscle homogenates were processed using the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Indianapolis, IN) on a BioMek FXP Laboratory Automation Workstation (Beckman Coulter, Indianapolis, IN) according to the manufacturer's instructions. The purity and concentration of the RNA samples were evaluated on a NanoDrop One/One spectrophotometer (ThermoFisher Scientific) and quantified using Quant-iT RiboGreen RNA Assay Kit (ThermoFisher Scientific). The RNA integrity was assessed with an RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, United States). RNA integrity numbers (RINs) for all samples were above 8.0 with a median RIN of 8.9.  mRNA libraries were prepared using the Universal Plus mRNA-Seq Library preparation kit (NuGEN). Briefly, mRNA was isolated from purified 400 ng total RNA using oligo-dT beads and used to synthesize cDNA following the NuGEN's instructions. The transcripts for ribosomal RNA (rRNA) and globin were further depleted using the AnyDeplete kit (NuGEN) prior to the amplification of libraries except for a subset of 15 sedentary muscle samples. The purified libraries were quantified using the Qubit dsDNA HS Kit (ThermoFisher Scientific). Library size and distribution were determined by Bioanalyzer using the High Sensitivity DNA kit (Agilent) or by Fragment Analyzer using High Sensitivity NGS Analysis kit (Agilent).
attributes.tag: GEO Accession
attributes.value: GSM4565666