design: RNA Preprocessing: Total RNA is first extracted, and its integrity is assessed using RNA-specific agarose gel electrophoresis or the Agilent 2100 Bioanalyzer, while RNA concentration and purity are measured. rRNA is then removed using a specialized kit, followed by fragmentation of RNA into 200-300bp segments via ion shearing. cDNA Synthesis: First-strand cDNA is synthesized using random primers during reverse transcription. For second-strand synthesis, a strand-specific library construction strategy is employed, where dUTP replaces dTTP to enhance the accuracy of subsequent analyses. Library Enrichment and Quality Control: PCR is performed to selectively enrich library fragments with adapters ligated at both ends. After enrichment, the library fragment size (target range: 300-1000bp) is verified using the Agilent 2100 Bioanalyzer, and the total library concentration is quantified via fluorescence-based methods to ensure the library meets sequencing requirements.