title: T.hyalina_400_t0_6
organism: Thalassiosira hyalina
description: Protocols: RNA sampling - Cultures of T. hyalina and N. frigida (n≥3) were concentrated by gentle vacuum filtration over 10 and 2 µm polycarbonate filters (Millipore), respectively. The concentrated samples were transferred to 2 ml reaction tubes and centrifuged at 4000 g for 10 min at 2°C in a microcentrifuge (Hettich; Bäch, Switzerland). The supernatant was removed, the cell pellet was flash frozen in liquid nitrogen and reaction tubes were stored at −80°C. Cultivation - Cells of T. hyalina (isolated in Svalbard in 2014, Norway by Klara Wolf) and N. frigida (isolated at Barrow, Alaska in May 2011 by Andrew Juhl), were grown in Arctic seawater that was supplemented with vitamins and trace metals according to the F/2 recipe (Guillard & Ryther, 1962) as well as macronutrients (~100 µmol L-1 nitrate; ~6 µmol L-1 phosphate). Media pCO2 was adjusted by aeration and checked with a handheld pH-meter (pH926 with an Aquatrode plus; Metrohm). In these media, cells were acclimated for >10 generations to the low-light regime of 20 µmol photons m-2 s-1. After this time, cells were sampled (t0) and light intensity was increased to 380 µmol photons m-2 s-1, resembling an upwelling event or ice break-up event, respectively. Cultures were sampled over the course of the following days after 2h, 24h and 120h, the last point reflecting a putative re-acclimated state. Differential gene expression (DGE) - Total RNA was extracted from samples (n≥3) after bead lysis of cells and on-column digestion of genomic DNA (DNAse I, 7 units, 30 min at room temperature) using the RNEasy Mini Kit (Macherey Nagel). Integrity and concentration of total RNA were verified using RNA6000 Lab Chips (Agilent). 1 µg of RNA was used for cDNA library preparation (TruSeq HT paired end kit, Illumina) and the success of library preparation was verified with a LabChip GX electrophoresis (Perkin Elmer). Libraries were adjusted in concentrations and pooled, yielding a 4 pM pool. The pool was spiked with PhiX standard as benchmark control, according to manufacturer´s protocol and the mixture was submitted for sequencing in an High Output FlowCell V2 (Illumina) using a NextSeq 500 sequencer (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-10-10T17:02:01Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-07-11T15:41:35Z
attributes.tag: External Id
attributes.value: SAMEA4786392
attributes.tag: INSDC center name
attributes.value: Alfred-Wegener-Institute, Helmholtz-Centre for Polar and Marine Research
attributes.tag: INSDC first public
attributes.value: 2018-10-10T17:02:01Z
attributes.tag: INSDC last update
attributes.value: 2018-07-11T15:41:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6999:T.hyalina_400_t0_6
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: Low pCO2
attributes.tag: sample name
attributes.value: E-MTAB-6999:T.hyalina_400_t0_6
attributes.tag: sampling site
attributes.value: Svalbard, Norway (2014)
attributes.tag: scientific_name
attributes.value: Thalassiosira hyalina