RNA-seq of Chionoecetes opilio: adult male hepatopancreas: exposed to chronic             noise
Total RNA was extracted from snow crab (Chionoecetes opilioh)                 hepatopancreas using TRIzol Reagent (Invitrogen), further purified using the                 phenol-chloroform phase separation method, treated with DNAse I (QIAGEN) and                 column-purified using the RNeasy Mini Kit (QIAGEN). Total RNA quantity and purity                 were assessed using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies)                 and its integrity was assessed using both 1% agarose gel electrophoresis and the                 2100 Bioanalyzer system (Agilent Technologies). Libraries were generated from 250 ng                 of total RNA. Briefly, mRNA enrichment was performed using the NEBNext Poly(A)                 Magnetic Isolation Module (New England BioLabs). cDNA was synthesized using the                 NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand                 Synthesis Modules (New England BioLabs). Library preparation was then completed                 using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs).                 Adapters and PCR primers were purchased from New England BioLabs. Libraries were                 quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) and the                 Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems).                 Average size fragment was determined using the LabChip GX (PerkinElmer). The                 libraries were normalized and pooled, denatured in 0.05N NaOH and then neutralized                 using HT1 buffer. ExAMP was added to the mix following the manufacturers                 instructions. The pool was loaded at 200pM on a cBot (Illumina) and the flowcell was                 run on a HiSeq 4000 (Illumina) for 2x100 cycles (paired-end mode). A phiX library                 was used as a control and mixed with libraries at the 1% level. The Illumina control                 software was HCS HD 3.4.0.38 and the real-time analysis program was RTA v. 2.7.7.                 The program bcl2fastq2 v2.18 was used to demultiplex the samples and generate fastq                 reads.