title: RNA-Seq of Pinellia ternata
design: This study enriched mRNA from total RNA using Oligo(dT) magnetic beads to capture polyadenylated transcripts. RNA fragmentation was achieved through ion-based shearing, producing approximately 300 base pairs (bp) fragments. The choice of 300 bp as the fragment length was based on the following considerations: shorter fragments would result in an overrepresentation of adapter sequences, thus reducing the propor-tion of valid data. In comparison, longer fragments could hinder efficient cluster for-mation during sequencing. cDNA synthesis was carried out using RNA as a template, with a six-base random primer and reverse transcriptase for the first-strand synthesis. The second strand of cDNA was then synthesized using the first strand as a template. Following cDNA synthesis, PCR constructed and amplified the library to enrich for de-sired fragment sizes. Library selection was performed based on fragment size, with a target size of 450 bp. The quality of the library was assessed using an Agilent 2100 Bi-oanalyzer, and both the library's total concentration and effective concentration were measured. Each was labeled with a unique index sequence to distinguish samples; the libraries were then pooled proportionally to their required sequencing depth. The pooled libraries were diluted to 2 nM and denatured by alkali treatment to generate single-stranded libraries. After RNA extraction, purification, and library construction, the samples were subjected to second-generation sequencing (Next-Generation Se-quencing, NGS) on the Illumina platform, utilizing paired-end (PE) sequencing tech-nology.