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).