GSM9136936: liver, RYGB chow, 4; Mus musculus; RNA-Seq
Total RNA extraction was performed using Trizol reagent (Ambion, Life Technologies, Carlsbad, CA), followed by purification and concentration using RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany; #74204). RNA purity was assessed by 260/280 and 260/230 ratios and samples were sent for sequencing. mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads, then fragmented using divalent cations at elevated temperature. First-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase, followed by second-strand synthesis using DNA Polymerase I and RNase H with dUTP incorporation to maintain strand specificity. After end repair, A-tailing, and adapter ligation, cDNA fragments were size-selected and amplified. Library quality and concentration were assessed using an Agilent Bioanalyzer, Qubit Fluorometer, and qPCR.