title: mutant
design: Ribosomal RNA (rRNA) was depleted from qualified total RNA samples using a prokaryote-specific rRNA removal kit to enrich messenger RNA (mRNA) and non-coding RNAs. The purified rRNA-depleted RNA was fragmented under optimized conditions. First-strand cDNA synthesis was performed using random hexamer primers. To enable strand-of-origin determination, dUTP was incorporated during second-strand cDNA synthesis instead of dTTP. The resulting double-stranded cDNA underwent end repair, 3'-end adenylation (A-tailing), and ligation of Illumina-compatible adapters. Adapter-ligated fragments within the target size range (typically 250-450 bp) were size-selected. Uracil-containing second strands were enzymatically digested using USER enzyme (NEB) to generate strand-specific libraries. The libraries were then amplified with index primers via limited-cycle PCR and purified. Final library concentration was quantified using Qubit fluorometry and qPCR, while fragment size distribution was verified using an Agilent Bioanalyzer. Libraries passing quality control (appropriate size distribution, absence of adapter dimers, sufficient concentration) were pooled in equimolar ratios based on qPCR-derived effective concentrations to achieve uniform sequencing depth across samples during paired-end sequencing (2x150 bp) on an Illumina NovaSeq 6000 platform.