GSM9421911: Patient_1_Primary_tumor _LRS; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIeasyTM Total RNA Extraction Reagent according to the manufacturer's instruction. LRS:Using the Iso-Seq Express 2.0 kit, polyadenylated RNA molecules were enriched with Oligo(dT) primers and reverse-transcribed to synthesize cDNA, followed by PCR amplification to generate barcoded cDNA. The Kinnex Full-Length RNA kit was then used to amplify the cDNA and introduce sticky ends, allowing concatenation of eight cDNA fragments in series while ligating barcoded adapters at both ends. Damage repair was performed, and exonuclease treatment was applied to remove incompletely ligated fragments, yielding the final sequencing library. After quantification, the library templates of a certain concentration and volume and enzyme complexes were loaded into the nanopores of the PacBio Revio sequencing system for single-molecule real-time (SMRT) sequencing. SRS:Eukaryotic mRNA was enriched using Oligo(dT) magnetic beads. The enriched mRNA was then fragmented using fragmentation buffer. Using the fragmented mRNA as a template, first- and second-strand cDNA were synthesized and subsequently purified. The purified double-stranded cDNA underwent end repair, A-tailing, adapter ligation, and fragment size selection to obtain cDNA fragments of approximately 350 bp. Finally, PCR amplification was performed to generate the cDNA library.The double-stranded cDNA was then denatured at high temperature to produce single-stranded DNA, followed by the addition of a circularization primer to form a single-stranded circular library. The constructed libraries were quality-checked using Qubit 3.0 and Agilent 2100 systems. Libraries that passed quality assessment were sequenced on a high-throughput sequencing platform with a PE150 strategy.