Full-Length Transcriptome Sequencing

The main process of library construction was as follows: The Clonetech SMARTerTM PCR cDNA Synthesis Kit was used to synthesize full-length cDNA of mRNA. Primer with Oligo dT was used for A-T base pairing with the polyA tail at the 3’ terminal of mRNA as primer for reverse synthesis of cDNA, and primer was added to the terminal of full-length cDNA synthesized in reverse. Full-length cDNA was obtained using PCR, PB magnetic beads were used to purify the amplified full-length cDNA, and small fragments of cDNA less than 1 kb were removed. The terminus of the full-length cDNA was repaired and connected to the SMRT dumbbell adapter. The fragments that were not connected to the adaptor were digested by exonuclease. PB magnetic beads were used to purify the fragments, and the sequencing library was obtained. After library construction, Qubit 3.0 was used for accurate quantification, and Agilent 2100 was used to detect the size of the library. After qualified detection, a PacBio sequencer was used to perform full-length transcriptome sequencing. The raw sequence generated by the PacBio sequencer totaled 86.4 Gbp and was deposited into the NCBI Sequence Read Archive (SRA) with accession number SRR22263802.