PacBio library preparation, sequencing and data analysis

RNA at OD260/280 > 2.0, OD260/230 at 1.8–2.1 and integrity number > 8 was selected for subsequent studies. The Iso-Seq library was prepared using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences (PN 100–092–800-03). Sequencing was performed on a Pacbio Sequel instrument. Sequence data were processed using the SMRTlink 5.1 software. The subreads sequence was obtained by the correction between subreads. According to whether the sequence contained 5’end primer, 3’end primer and polyA tail, the sequences were divided into full-length sequence and non-full-length sequence. Isoform level clustering was used to cluster the full-length sequence to obtain the Cluster consensus sequence; finally, the non-full-length sequence was used for polishing the obtained consensus sequence to obtain high-quality sequences for subsequent analysis [54]. Any redundancy in corrected consensus reads was removed by CD-HITv4.6 package (−c 0.95 -T 6 -G 0 -aL 0.00 -aS 0.99) to obtain final transcripts for the subsequent analysis.