2.10 RNA-Seq and bioinformatics analysis
Pancreatic cancer cells were plated in six-well plates. After overnight incubation, the cells were treated with different concentrations of TA for 24 h. Total RNA was prepared as mentioned above. A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were constructed with the VAHTS mRNA-seq v2 Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s instructions, and index codes were added to attribute sequences to each sample. Transcriptomic sequencing was performed by using the Illumina NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA) with three biological replicates, and 150-bp paired-end reads were generated. The differentially expressed genes (DEGs) between treated and untreated groups were identified by using the DEGSeq R package (1.20.0). Gene ontology (GO) analysis of DEGs was performed using the Gene Ontology Annotation (GOA) database (http://www.ebi.ac.uk/GOA). KEGG pathway analysis of DEGs was conducted using the KEGG Automatic Annotation Server (KAAS; http://www.genome.jp/tools/kaas/).