title: Illumina HiSeq 2500 paired end sequencing; Seasonal RNAseq of Antarctic krill, Euphausia superba, from three different latitudinal regions of the Southern Ocean
design: Seasonal RNAseq of Antarctic krill, Euphausia superba, from three different latitudinal regions of the Southern Ocean
library_construction_protocol: Antarctic krill samples (Euphausia superba) were obtained from five different expeditions and from a Norwegian fishing vessel.  Sampling was carried out with a Rectangular Midwater Trawl (RMT8+1 for expeditions ANT23-2, ANT23-6 and JR15004, RMT8 for expedition JR260B), an Isaacs-Kidd Midwater Trawl (IKMT, expedition AMLR14) and a continuous pumping system (Norwegian fishing vessel).  Snap-frozen Antarctic krill samples stored at -80°C were transferred to the Alfred-Wegener-Institute, Bremerhaven, for molecular analysis.The Antarctic krill originated from three different latitudinal regions: a) Lazarev Sea (62°S -66°S), b) South Orkneys/Bransfield Strait (60°S-63°S), and c) South Georgia (54°S), including summer and winter samples for each region. By visual inspection of the outer sexual organs, male petasma and female thelycum, adult males and females were identified. RNA extraction was performed from frozen krill heads with the RNeasy Midi Kit (QIAGEN, Hilden, Germany). Frozen krill heads were cut on dry ice and moved to 1.5 mL RLT lysis buffer in tissue homogenizing Precellys® tubes (CKMix Tissue Homogenizing Kit, Bertin corp., Rockville, MD, USA). Homogenization was carried out at 4°C in a Precellys® homogenisator with the Cryolys® cooling system (Bertin corp.) with two runs for 15 s at 5000 rpm and 10 s break.  Further steps of RNA extraction were carried out according to the manufacturer's protocol of the RNeasy Midi Kit. The quality and quantity of the RNA was inspected using the NanoDrop™2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the Agilent 2100 Bioanalyzer system (Agilent technologies, Santa Clara, CA, USA). RNA samples were sent for sequencing to IGA Technology Services, Udine, Italy. Library preparation was performed with 1-2 μg RNA using the TruSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, CA, USA) after manufacturer's instructions. The poly-A mRNA was fragmented 3 minutes at 94°C. 1X Agencourt AMPure XP beads (Agencourt Bioscience Cooperation, Beckman Coulter, Beverly, MA, USA) were used for every purification step. The RNA samples and final libraries were quantified with the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quality tested by Agilent 2100 Bioanalyzer Nano assay.  For cluster generation on the flow cell, libraries were processed with cBot (Illumina, San Diego, CA, USA) following manufacturer's instructions.