2.2. RNA extraction and compete transcript sequencing
Total RNA was extracted from the mixed samples using Phygene Total RNA Isolation Kit (Phygene, Fuzhou, Fujian, China) following the manufacturer’s instructions. RNA purity was determined firstly by agarose gel electrophoresis, followed by use of a NanoDrop instrument (IMPLEN, CA, USA). RNA quantity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The total RNA from three samples was used to generate one library. The poly (A) mRNA was isolated via Oligo (dT), and the mRNA was reversed into full length first-strand cDNA using a SMARTer PCR cDNA Synthesis Kit. The synthesized cDNA was enriched using PCR amplification. Fragment screening of partial cDNAs were performed by the BluePippin Size Selection System, and the 5-10 kb size optional fragments were selected and subsequently enriched using PCR amplification. The amplicators were used to construct a SMRTbell Library, subsequently sequenced on the PacBio RS II sequencing platform (HiSeq 2500, San Diego, CA, USA), and final complete reads were generated.