RNA-Seq analysis

The obtained cDNA library was then sequenced on the Illumina platform. The raw data was filtered using Trimmomatic, resulting in clean data. Reads were aligned to the reference genome with HISAT2 (version: 2.2.1). For three biological replicates, DESeq2 (version: 1.34.0) was used to identify differentially expressed genes, while edgeR (version: 3.36.0, dispersion = 0.01) was applied for datasets without replicates. We first selected differentially expressed genes with a Fold Change ≥ 2 and FDR < 0.05. If the number of differentially expressed genes in each comparison group exceeded 1,000, we tightened the selection criteria to Fold Change ≥ 2 and FDR < 0.01. In this analysis, we ultimately chose to filter differentially expressed genes with an FDR < 0.01. Differentially expressed genes and samples were clustered using the R package Pheatmap. StringTie was used to map reads, assemble transcripts, and compare them with known genes using Cuffcompare. Differential splicing was analyzed with rMATS. Finally, Samtools aligned the sorted, PCR-deduplicated files with the reference sequence to detect variants. In the analysis data, abbreviation "B" represents BTZ treatment, and "CB" represents CURE treatment.