RNA-sequencing assay to Identify changed gene expression in stomach adenocarcinoma  (STAD) cells with ACAT2 knockdown
Purpose: To elucidate how ACAT2 influenced the expression profile in STAD cells, ACAT2 expression was reduced using one shRNA in STAD HGC-27 cells. Then, transcriptional profiling of cells with ACAT2 knockdown (shACAT2) and the control cells (shNC) was performed to characterize differentially expressed genes (DEG).Methods: When shNC and shACAT2 cells grew to approximately 80% confluence, these cells were washed twice using pre-chilled PBS. Total RNAs were harvested using Trizol reagents (Thermo Fisher Scientific).  Poly(A) mRNA was isolated and enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module. The mRNA was then recovered for library generation with NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, E7420S) following the manufacturer's instructions. The cDNA libraries were sequenced at WuXiNextCODE (China). FastQC was used to examine the quality of raw reads. Read alignment was conducted using STAR (v2.5.1b),  and R package edgeR (v3.8.5) was used to determine relative transcript abundances and differentially expressed genes (DEGs) between sample pairs. Results: Generally, 241 genes  were increased and 117 genes decreased in HGC-27 shACAT2 cells, compared with shNC cells (FDR<0.05, Fold Change>2). GO analysis showed that these changed genes were significantly enriched in signal pathways related to cancer. Conclusions: Reduced ACAT2 expression alters multiple cancer-related signal pathways. Overall design: Three biological replicates for each group shNC and shACAT2 - were subjected to RNA-sequencing analysis respectively, among which shNC acted as the reference sample for shACAT2.