2.7. RNA Sequencing (RNA-Seq) and Data Analysis

Total RNA was extracted from the resected tumors of the PBS, OXP, DAC, and DAC plus OXP groups on day 5 (1 day after completing DAC treatment) by using the TRIzol reagent. Message RNA was extracted from the total RNA and cut into short fragments with ~200 bases as templates for cDNA synthesis. The cDNAs subsequently used to establish a cDNA library by PCR amplification and sequenced by using an Illumina HiSeqTM 2500 platform [31]. Clean reads were obtained by trimming the adaptor sequences from raw reads, and these reads were then were used for further transcript annotation and calculation bases on the fragments per kilobase per million reads (FPKM) method. Differential gene expressions (DEGs) were identified with the DESeq software package. The Benjamini–Hochberg false discovery rate was employed to correct the p values with a significant level set at 0.05 [31].