RNA Extraction and RNA-seq Library Preparation

Total RNA was extracted using a Trizol reagent (Transgen Biotech, Beijing, China). Thermo Scientific NanoDrop 2000c (ThermoFisher Scientific Inc., Waltham, MA, USA) was used to determine the concentration and purity of the extracted RNA. The integrity of the RNA was detected by 1% agarose gel electrophoresis. The total amount of RNA should be ≥1 μg, the concentration ≥ 50 ng/μL, and the value of OD 260/OD 280 should be between 1.8 and 2.2 [20 (link)]. An oligomer magnetic bead (dT) approach was used to enrich Poly A mRNA from total RNA (6 samples) [21 (link),22 (link)]. A cDNA library was constructed by synthesizing cDNA from random hexamers, purifying cDNA, and amplifying it by PCR. A NEBNext^® Ultra RNA Library Prep Kit for Illumina was used to perform RNA-seq library preparation and, after library inspection, qualified. Finally, paired-end sequencing of different libraries was accomplished using Illumina sequencing.