4.4. RNA Extraction and Library Construction

Total RNA was isolated using Trizol reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions. RNA purity was tested using a NanoDrop 2000 (Thermo, Waltham, MA, USA), RNA concentration was measured using an Agilent 2100 Bioanalyzer, and RNA integrity was evaluated with an Agilent RNA6000 NanoKit according to the manufacturer’s protocol.

Each sample required approximately 4 to 8 μg of total RNA to construct the RNA-Seq libraries. The method consisted mainly of the following steps: (1) mRNA was first purified from total RNA using polyA magnetic bead enrichment. (2) Chemical fragmentation was conducted. (3) Random hexamer priming was used to convert mRNA into first single-strand cDNA and to synthesize second-strand cDNA. (4) Sequencing adaptors were added to cDNA fragments. Suitable fragments were selected based on the separation by agarose gel electrophoresis. (5) PCR amplification construction of RNA libraries was completed. In total, 24 RNA libraries were constructed, and the quality and quantity of each RNA library were assessed using Nanodrop ND-1000 spectroscopy and an Agilent 2100 Bioanalyzer according to the protocol guide.