design: The quality and integrity of the initial total RNA were first assessed. Subsequently, messenger RNA (mRNA) containing polyadenylated tails was isolated from the total RNA pool using oligo(dT)-attached magnetic beads. The enriched mRNA was then chemically fragmented into smaller pieces. First-strand cDNA was synthesized from the fragmented mRNA templates using reverse transcriptase and random hexamer primers. The second-strand cDNA was then synthesized using DNA polymerase I, incorporating deoxyuridine triphosphate (dUTP) in place of deoxythymidine triphosphate (dTTP). The resulting double-stranded cDNA fragments were end-repaired, A-tailed, and ligated to Illumina sequencing adapters. Prior to PCR amplification, the dUTP-marked second strand was digested by Uracil-DNA Glycosylase (UDG), ensuring that only the first-strand cDNA, which reflects the original mRNA orientation, was amplified. This process resulted in a strand-specific library ready for paired-end sequencing.