RNA-seq of muscle: female Tachypleus tridentatus
We purified mRNA by depleting rRNA from total RNA using the RNA Purification Beads. The remaining RNA was cleaned three times using Beads Binding Buffer, and eluted RNA was incubated at 94C for 8 min. First-strand cDNA synthesis was performed by combining the fragmented mRNA with 8 l of First Strand Synthesis Act D Mix and SuperScript II Reverse Transcriptase and incubated at 25C for 50 min, dropped to 4C. For second strand synthesis, we added 5 l of End Repair Control and 20 l of Second Strand Marking Master Mix and incubated at 16C for 60 min. The reaction was then purified using 90 l of AMPure XP beads. Subsequently, A-Tailing Control and Ligation Control were applied to A-tailing and adapter ligation of the double stranded cDNA, respectively. In the cDNA fragments enrich reaction process, we added 5 l of PCR Primer Cocktail and 25 l of PCR Master Mix and ran the initial denaturing at 98C for 30 sec, followed by 15 cycles of denaturing at 98C for 10 sec, annealing at 60C for 30 sec, and extending at 72C for 30 sec, and finished with a final extending at 72C for 5 min. RNA-seq libraries were diluted to 10 pM and quantified using an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina HiSeq 2000 across one lane with paired-end 150 bp.