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.