Southern Ocean Experimental Metatranscriptome to Investigate Micronutrient Colimitation

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Como citar

Pesquisadores deveriam citar esta obra da seguinte maneira:

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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O editor e o detentor dos direitos deste trabalho é SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY 4.0) License.

GBIF Registration

Este recurso foi registrado no GBIF e atribuído ao seguinte GBIF UUID: ddd527ae-1539-4189-ac4a-a731640badcf.  SCAR - Microbial Antarctic Resource System publica este recurso, e está registrado no GBIF como um publicador de dados aprovado por Scientific Committee on Antarctic Research.

Palavras-chave

Metadata

Contatos

Cobertura Geográfica

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

Cobertura Taxonômica

RNA metatranscriptome

Cobertura Temporal

Dados Sobre o Projeto

Nenhuma descrição disponível

O pessoal envolvido no projeto:

Métodos de Amostragem

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.

Descrição dos passos do método:

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.

Citações bibliográficas

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.

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