Wedelolide A induces apoptosis, autophagy disruption, and the System Xc--GPX4 pathway-mediated ferroptosis in gastric cancer cells through ROS

After treating the MGC-803 xenograft nude mice with WA for 19 days, the mice were anesthetized and sacrificed, and the tumors were extracted for RNA-Seq. Briefly, the tumor samples were prepared for total RNA extraction, mRNA isolation and purification, sequencing library preparation, and sequencing. Next, total RNA was extracted from the tissue using TRIzol Reagent according the manufacturer s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Messenger RNA was used to synthesize double-stranded cDNA with the SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers. The library was PCR amplified for 15 cycles using Phusion DNA polymerase (NEB). After quantification with Qubit 4.0, the sequencing library was performed on the NovaSeq X Plus platform (PE150) using the NovaSeq Reagent Kit. The raw paired end reads were trimmed and quality controlled by fastp with default parameters. Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 software. The mapped reads of each sample were assembled by StringTie in a reference-based approach. The data were analyzed on the online platform of Majorbio Cloud Platform.