title: IsoSeq_root
design: Iso-Seq libraries were prepared with the SMRTbell Prep Kit 3.0 according to the prescribed method. Total RNA was isolated and mRNA was enriched. The mRNA was then reverse-transcribed into cDNA using an oligo-dT primer. Double-stranded cDNA was synthesized and subjected to damage repair and end-conditioning to create blunt-ended, 5'-phosphorylated ends. SMRTbell adapters were ligated to the prepared cDNA, forming the SMRTbell template required for sequencing. Exonuclease treatment was applied to remove failed ligation products and to ensure that only SMRTbell templates were present. The library was then size-selected to the desired range using the BluePippin Size Selection System. Following size selection, the library was assessed for quality and quantity. Once validated, the library was sequenced on a PacBio Sequel II system, which employed Single Molecule, Real-Time (SMRT) technology to generate long reads for full-length transcript sequencing.