Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Briefly, cells were lysed in TRIzol, and RNA was separated with chloroform, precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. RNA quantity and quality were assessed using NanoDrop and Agilent Bioanalyzer. Library preparation was conducted using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions