Sequencing adapters were then ligated to both ends of the double-stranded cDNA, enabling identification and sequencing during downstream processes. The adapter-ligated fragments were purified and size-selected using DNA Clean Beads to enrich for the desired library fragments. Next, PCR amplification was carried out with P5 and P7 primers to generate the final library. UDG (Uracil-DNA Glycosylase) was included in the PCR mix to degrade the second strand containing dUTP, ensuring strand specificity.