RAW264.7 macrophage cells treated with 30 ng/mL mouse SAA1 repeat 3 (CRX2028210; CRA030548)
Total RNA was enriched for messenger RNA using poly-T oligo-attached magnetic beads and subsequently fragmented into short pieces. First-strand cDNA was synthesized with random hexamer primers, followed by second-strand synthesis in which dUTP was incorporated instead of dTTP to retain strand information. The resulting cDNA underwent end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, PCR amplification, and purification to generate strand-specific libraries. Library quality and concentration were assessed using Qubit fluorometry, real-time PCR, and an Agilent Bioanalyzer.