Smrt seq

SMRTlink 6.0 software was used to process the PacBio SMRT-seq raw reads. The CCS was obtained from the subreads.bam file. Sequencing adapters were trimmed, then clean CCS were classified into either full- or non-full-length isoforms based on cDNA primers and poly-A tail signal. To improve consensus accuracy, Iterative Clustering for Error Correction (ICE) and Arrow algorithm (https://downloads.pacbcloud.com/public/software/installers/smrtlink_5.0.1.9585.zip) were used to obtain high quality isoforms and full-length sequences. Additional nucleotide errors in consensus reads were corrected using the Illumina RNA-seq data with the software LoRDEC (Helsinki, Finland) [86 (link)]. BUSCO [87 (link)] was used to explore completeness according to conserved ortholog content.

Total RNA was extracted with TRIzol reagent (Life Technologies, USA) according to the manufacturer’s instructions. RNA quality was assessed by a 1% agarose gel and the concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity was examined using the Agilent Technologies 2100 Bioanalyzer system (Santa Clara, CA) with a RNA integrity number cutoff greater than 7.
To obtain the complete information of all transcripts, SMRT-seq (PacBio) was applied in this study. The best RNA sample (2 populations × 3 replicated samples) was selected and then pooled together in equal quantity for SMRT-seq. Full-length cDNA was synthesized using the SMRTer PCR cDNA Synthesis Kit (Biomarker, Beijing). PacBio Sequel II sequencing reactions from one SMRT cell (1–6 kb) were performed.
For Illumina RNA-seq, six RNA samples (2 populations × 3 replicated samples) were used. Then, the six libraries for sequencing were generated using NEBNext® Ultra™ RNA Library Prep Kit (NEB, Beverly, MA, USA) according to the manufacturer’s recommendations. Subsequently, the prepared libraries were sequenced on an Illumina NovaSeq 6000 platform (2 × 150 bp).