design: The standard RNA-seq library preparation begins with the assessment of RNA integrity, followed by either poly-A selection (to capture mRNA) or rRNA depletion (to remove ribosomal RNA). The enriched RNA is then fragmented into smaller pieces using heat and divalent cations, which serve as templates for first-strand cDNA synthesis using reverse transcriptase and random primers. Subsequently, the second strand is synthesized, and the resulting double-stranded cDNA undergoes end-repair and dA-tailing to facilitate the ligation of sequence-specific adapters. These adapters contain barcodes (indices) for multiplexing and sequences compatible with the flow cell. Finally, the library is amplified via PCR to enrich for fragments with successful adapter ligation and purified using magnetic beads, resulting in a final library ready for high-throughput sequencing.