design: RNA-seq of UOX-KO Mouse Liver
library_construction_protocol: Mice were euthanized by cervical dislocation at 12 weeks of age. The liver tissue was immediately dissected, rapidly frozen in liquid nitrogen, and subsequently stored at -80°C. Animal samples were extracted using the Trizol method. The extracted RNA was dissolved in 50 μL of DEPC-treated water. The quantity and quality of the total RNA were assessed using a Qubit fluorometer and a Qsep400 high-throughput biofragment analyzer. Strand-specific RNA-seq libraries were prepared. Briefly, mRNA was enriched from total RNA using Oligo(dT) magnetic beads. The purified mRNA was fragmented using fragmentation buffer at an elevated temperature. First-strand cDNA was synthesized using random hexamer primers. Second-strand cDNA was synthesized incorporating dUTP in place of dTTP to achieve strand specificity, followed by end repair, A-tailing, and adapter ligation. Libraries were size-selected for insert sizes of 250-350 bp using DNA magnetic beads and then amplified by PCR. Final library quality was assessed using a Qubit fluorometer and a Qsep400 analyzer. For the DNB platform, the double-stranded DNA library was circularized to form single-stranded circular DNA, which was then rolled-circle amplified to produce DNA nanoballs (DNBs).