Southern Ocean Experimental Metatranscriptome to Investigate Micronutrient Colimitation

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Les chercheurs doivent citer cette ressource comme suit:

Bertrand E, McCrow J, Moustafa A, Zheng H, McQuaid J, Delmont T, Post A, Sipler R, Spackeen J, Xu K, Bronk D, Hutchins D, Allen A (2019): Southern Ocean Experimental Metatranscriptome to Investigate Micronutrient Colimitation. v1.2. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=southern_ocean_experimental_metatranscriptome&v=1.2

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L’éditeur et détenteur des droits de cette ressource est SCAR - Microbial Antarctic Resource System. Ce travail est sous licence Creative Commons Attribution (CC-BY) 4.0.

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Cette ressource a été enregistrée sur le portail GBIF, et possède l'UUID GBIF suivante : ddd527ae-1539-4189-ac4a-a731640badcf.  SCAR - Microbial Antarctic Resource System publie cette ressource, et est enregistré dans le GBIF comme éditeur de données avec l'approbation du Scientific Committee on Antarctic Research.

Mots-clé

Metadata

Contacts

Couverture géographique

Souther Ocean sea water of the ice edge, near McMurdo Sound, Antarctica

Couverture taxonomique

RNA metatranscriptome

Couverture temporelle

Données sur le projet

Pas de description disponible

Les personnes impliquées dans le projet:

Méthodes d'échantillonnage

Water was pumped to the surface using a trace metal clean diaphragm pump and acid cleaned teflon tubing and dispensed into trace metal clean (TMC) 50 L carboys. Sampling occurred between 18:00 and 19:00 in open air, with wind coming from over open water, NNE to NE. The carboys were protected from light with dark plastic bags and returned to the Crary Laboratory at McMurdo Station via helicopter within one hour of sampling, where it was stored overnight at 0°C and then split into twelve 2.7 L TMC polycarbonate bottles.

Description des étapes de la méthode:

1. Three bottles were left as unamended controls, three were amended with 1 nM FeCl3, three were amended with 200 pM cyanocobalamin, and three had 200 pM cyanocobalamin and 1 nM Fe added. Iron concentrations in the cobalamin stock were such that < 6 pM iron was added with 200 pM cobalamin (measured via flow injection). Bottles were placed in an indoor incubator at 0°C, ~45 μmol photons m-2 s-1 of constant light. This level of irradiance is in between levels typical for 3 m depth in open water and under the sea ice, and was selected because the harvested community would have experienced both under ice and open water light regimes in the recent past.
2. For RNA extraction, 1 billion copies of each of two RNA standards were added to each filter (#1, #8 ArrayControl Spots and Spikes, Life Technologies, with polyA tails). RNA was extracted from Sterivex filters using the Trizol reagent manufacturer’s protocol (Life Technologies); Trizol was added to the filter membrane after it had been extracted from the plastic housing on dry ice using a sterile pipe cutter, razor blade, and forceps. 450- 800 ng RNA was obtained per filter. RNAeasy MinElut Cleanup kit was applied (Qiagen) and ribosomal RNA was removed with Ribo-Zero Magnetic kits, employing a mix of plant, bacterial, and human/mouse/rat formulations in a ratio of 2:1:1 (Epicentre).
3. The resulting mRNA enrichment was purified using an Agencourt RNAClean XP kit and 2 ng of rRNA depleted RNA was subjected to amplification and cDNA synthesis using the Ovation RNA-Seq System V2 (NuGEN), which applies both polyA and random hexamer primers. 1 μg of the resulting high quality cDNA pool was fragmented to a mean length of 200 bp. Libraries were constructed using a Truseq RNA Sample Prep kit v2 (IlluminaTM), applying the manufacturer’s protocol from the end-repair step. The libraries were then subjected to paired-end sequencing via Illumina Hiseq.

Citations bibliographiques

1. Bertrand, E. M., McCrow, J. P., Moustafa, A., Zheng, H., McQuaid, J. B., Delmont, T. O., ... & Bronk, D. A. (2015). Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge. Proceedings of the National Academy of Sciences, 112(32), 9938-9943.

Métadonnées additionnelles