GSM8390259: WT sham, rep5; Mus musculus; RNA-Seq
Total RNA was purified using an RNeasy Mini Kit (Qiagen). The integrity of total RNA was assessed using Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) with the High Sensitivity RNA Analysis Kit. RNA Quality Numbers (RQNs) from 1 to 10 were assigned to each sample to indicate its integrity or quality. “10” stands for a perfect RNA sample without any degradation, whereas “1” marks a completely degraded sample. RNA samples with RQNs ranging from 8 to 10 were used for RNA library preparation with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Briefly, mRNA was isolated from 1-2.5 µg of total RNA using poly-T oligo attached to magnetic beads and then subjected to fragmentation, followed by cDNA synthesis, dA-tailing, adaptor ligation and PCR enrichment. The sizes of RNA libraries were assessed by Fragment Analyzer with the High Sensitivity NGS Fragment Analysis Kit. The concentrations of RNA libraries were measured using the StepOnePlus Real-Time PCR System (ThermoFisher Scientific, San Jose, CA) with the KAPA Library Quantification Kit (Kapabiosystems, Wilmington, MA).