RNA Extraction and RNA-Seq

Total RNA was extracted from the specimens using TRIzol (Life Technologies, Carlsbad, CA, United States) according to the manufacturer’s instructions. The RNA was qualified and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). After rRNAs were removed, the rest of RNAs were fragmented using a fragmentation buffer and reverse-transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP), and buffer. Next, the cDNA fragments were purified using a QIAquick PCR Extraction Kit (Qiagen, Hilden, Germany), end-repaired, polyadenylated, and ligated to Illumina sequencing adapters. Uracil-N-glycosylase was used to digest the second-strand cDNA. After PCR amplification, the digested products were purified using AMPure XP Beads and a High Sensitivity DNA Assay Kit (Agilent Technologies, United States) was used for library quality inspection. RNA sequencing was carried out by Gene Denovo Biotechnology Company (Guangzhou, China) using a HiSeq 2500 system (Illumina, San Diego, CA, United States).