Total RNA was isolated from cells using TRIzol (Invitrogen) following the manufacturer's instructions. RNA quality and integrity were evaluated on an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Kit, and only high-quality samples were taken for further analysis. Strand-specific, poly(A)-selected libraries were generated from 500 ng of total RNA using the KAPA Stranded mRNA Sample Preparation Kit. Briefly, mRNA was captured with poly-T–coated magnetic beads, fragmented, and converted into first-strand cDNA. Second-strand synthesis produced double-stranded cDNA, which was subsequently A-tailed and ligated to sequencing adapters (NEB). Adapter uracils were removed with USER enzyme prior to PCR amplification. Library size distribution (~300 bp) was assessed using the Agilent High Sensitivity DNA kit, and concentrations were measured using the QuantiFluor dsDNA system (Promega). Libraries were sequenced on an Illumina NovaSeq 6000 platform in 2 × 151 bp paired-end mode.