Sequencing, assembly and annotation of the transcriptome

After assessing the quality and quantity of RNA samples by using a Qubit 2.0 Fluorometer (Invitrogen Life Technologies, Waltham, MA, United States) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States), cDNA libraries were produced via NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB, United States), along with the manufacturer’s recommendations. The libraries were sequenced on an Illumina Hiseq–PE150 platform using a paired-end sequencing strategy. The initially generating sequences (raw reads) were filtered after removing adapter sequences, reads with poly-N and low quality reads. Next, the resulting reads (clean reads) were assembled into the trinity transcriptome using Trinity v2.5.1 (Grabherr et al., 2011), or used for subsequent analyses. After the transcripts were clustered by Corset v1.05 with default settings (Davidson and Oshlack, 2014), the longest transcript in each cluster was picked and pooled into a unigene database (defined as the unigene transcriptome). Using the unigenes as queries, we aligned them into seven functional databases (GO, Gene Ontology; KO, KEGG Ortholog; KOG/COG, EuKaryotic Orthologous Groups/Clusters of Orthologous Groups; NR, National Center for Biotechnology Information (NCBI) non-redundant (NR) protein sequences; NT, NCBI non-redundant nucleotide sequences; PFAM, Protein family and SwissProt). The annotated information of unigenes in each database was summarized.