RNA-seq of Chionoecetes opilio: adult male hepatopancreas: Lilly Canyon control site: 2d       after 2D seismic exposure at Carson Canyon
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.
SUB5028023
LCAS_106