CuYeXuanGouZi_ISO (CRX1848061; CRA027668)
In order to generate full-length transcriptome data, total RNA samples meeting quality criteria (RIN >= 7.0, input 300 ng) were subjected to mRNA enrichment via Oligo(dT) magnetic beads. The enriched mRNA was fragmented and reverse-transcribed using the Iso-Seq Express 2.0 Kit, which employs template-switching oligonucleotides (TSO) to generate full-length cDNA. PCR amplification was performed with barcoded ISO-Seq primers (bc01-bc12), and the products were purified using SMRTbell magnetic beads. Purified cDNA underwent eight parallel Kinnex PCR reactions with specific primer mixes to generate directionally fragmented DNA sequences. The amplified products were pooled and ligated to Kinnex barcoded adapters (bc01-bc04), forming linear arrays. Subsequent steps included DNA repair, nuclease treatment, and two rounds of SMRTbell bead purification. The final Kinnex library was adjusted to a concentration <60 ng/microL, validated for size distribution via the Femto Pulse system, and sequenced on the PacBio Revio platform. submitted_library_strategy: ISO-Seq submitted_instrument_model: PacBio Sequel