Male crab hepatopancreas under drought at room temperature for 30 hours
Total RNA was extracted from hepatopancreas using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number > 7.0. Poly (A) RNA was purified from 1 g total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA). Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA libraries using TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, USA). After quality control for raw sequencing reads, libraries were sequenced on an Illumina Hiseq 4000 (LC-Bio Technology CO., Ltd., Hangzhou, China) platform and 2150 bp paired-end reads were generated. Raw data were firstly subjected to quality control before bioinformatics analysis. And then, the screened high-quality reads were mapped to the E. sinensis genome (unpublish) using HISAT package (Kim et al., 2015). The mapped reads of each sample were assembled using StringTie (Pertea et al., 2015). Then, all transcriptomes from samples were merged to reconstruct a comprehensive transcriptome using perl scripts.