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/).