RNA-seq of gill: adult female Charybdis japonica
The total RNA of 9 individual C. japonicas were extracted using the standard Trizol Reagent Kit (Huayueyang Biotech Co., Ltd., Beijing, China) following the manufacturers protocol, respectively. The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was applied to quantify the each extracted total RNA. The mRNA was further purified by using RNA Purification Beads (Illumina, San Diego, CA, USA) to deplete rRNA from each total RNA, and then cleaned three times with Beads Binding Buffer (Illumina, San Diego, CA, USA). After incubating the mRNA for 8 min at 94C, fragmented mRNA was combined with 8 l of First Strand Synthesis Act D Mix (Illumina, San Diego, CA, USA) and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), and the conjugated product was incubated at 25C for 50 min to synthesize the first-strand cDNA. Furthermore, we added 5 l of End Repair Control (Illumina, San Diego, CA, USA) and 20 l of Second Strand Marking Master Mix (Illumina, San Diego, CA, USA) and incubated at 16C for 60 min to complete the synthesis of the second-strand synthesis. The reaction was then purified using 90 l of AMPure XP beads (Beckman Coulter, Beverly, USA). We then used A-Tailing Control and Ligation Control to add A-tailing and adapter ligation for each double-stranded cDNA. Subsequently, we enriched the cDNA fragment and the enrich reaction system as follows: 5 l of PCR Primer Cocktail (Beckman Coulter, Beverly, USA), 25 l of PCR Master Mix (Beckman Coulter, Beverly, USA), the initial denaturing at 98C for 30 sec, the 15 cycles of denaturing at 98C for 10 sec, the annealing at 60C for 30 sec, and the extension at 72C for 30 sec, the final extension at 72C for 5 min. All RNA-seq libraries were diluted to 10 pM and then quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Finally, sequencing of 9 RNA-seq libraries were performed on an Illumina HiSeq 2000 platform across one lane with 150 bp paired-end.