2.3 RNA extraction and sequencing
For the gene annotation and the prediction of protein-coding genes, 11 tissues above-mentioned of C. sonnerati were used to conduct transcriptome sequencing. Total RNA was extracted with the Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The concentration and integrity of total RNA were estimated using the Agilent 2100 Bioanalyzer (Aglient Technologies, USA) and ethidium bromide staining of 28S and 18S ribosomal bands on a 1% agarose gel, respectively. Equal volumes of RNA samples were pooled together for RNA library construction and sequencing. Briefly, the full-length cDNA was prepared using a SMARTerTM PCR cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). The SMRTbell libraries were constructed with the Pacific Biosciences DNA Template Prep Kit 2.0. Library. Library quantification and size were checked using a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and a 2100 Bioanalyzer system (Agilent Technologies, CA, USA), respectively. Subsequently, SMRT sequencing was carried out with a PacBio Sequel II platform by Frasergen Bioinformatics Co., Ltd. (Wuhan, China).