Marine_ microbial eukaryotes (18S) from the Ross Sea (Antarctica)

Última versión Publicado por SCAR - Microbial Antarctic Resource System en Mar 19, 2019 SCAR - Microbial Antarctic Resource System

Amplicon sequencing dataset of microbial eukaryotes (18S ssh rRNA gene v9) in water and sea ice collected in the Ross Sea, Antarctica during the summer of 2011. Prior to sequencing, mixotroph abundances were determined using tracer ingestion.

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¿Cómo referenciar?

Los usuarios deben citar este trabajo de la siguiente manera:

Gast R, Fay S, Sanders R (2018): Marine_ microbial eukaryotes (18S) from the Ross Sea (Antarctica). v1.2. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=marine_microbial_eukaryotes_18s_experiment_rosssea&v=1.2

Derechos

Los usuarios deben respetar los siguientes derechos de uso:

El publicador y propietario de los derechos de este trabajo es SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 License.

Registro GBIF

Este recurso ha sido registrado en GBIF con el siguiente UUID: b9c72483-b00a-4848-9239-b36dce06b0cb.  SCAR - Microbial Antarctic Resource System publica este recurso, y está registrado en GBIF como un publicador de datos avalado por Scientific Committee on Antarctic Research.

Palabras Clave

Metadata

Contactos

¿Quién creó el recurso?:

Rebecca Gast
Woods Hole Oceanographic Institution Woods Hole US
Scott Fay
Temple University Philadelphia US
Robert Sanders
Temple University Phyladelphia US

¿Quién puede resolver dudas acerca del recurso?:

Rebecca Gast
Woods Hole Oceanographic Institution Woods Hole US

¿Quién documentó los metadatos?:

Maxime Sweetlove
rResearch assistent
Royal Belgian Institute for Natural Sciences Rue Vautier 29 1000 Brussels

¿Quién más está asociado con el recurso?:

Usuario

Cobertura Geográfica

Southern Ocean: Ross Sea, Antarctica

Coordenadas límite Latitud Mínima Longitud Mínima [-75.44, -171.29], Latitud Máxima Longitud Máxima [-72.35, 178.3]

Cobertura Taxonómica

Microbial eukaryotes (18S ssh rRNA gene, v9 region)

Dominio  Eukaryote (Eukaryotes)

Cobertura Temporal

Fecha Inicial / Fecha Final 2011-01-26 / 2011-02-08

Datos del Proyecto

No hay descripción disponible

Título National Science Foundation Grants OPP-0838955 (RG) and OPP-0838847 (RS)
Fuentes de Financiación This work was supported by National Science Foundation Grants OPP-0838955 (RG) and OPP-0838847 (RS).

Personas asociadas al proyecto:

Rebecca Gast

Métodos de Muestreo

Seawater was collected from two depths (surface and deep chlorophyll maximum) at 10 open water stations using 30 liter Niskin bottles on a CTD rosette. Two of those sites were sampled a second time on different dates. The deep chlorophyll maximum (CM) was determined by fluorescence (WetLabs FLRTD fluorometer) during the downcast of the CTD and ranged from 30 to 90 m in depth depending on station.

Área de Estudio Samples for shipboard experiments were collected in the Ross Sea during research cruise NBP-1101 aboard the R/V Nathaniel B. Palmer in the austral summer (20 January through 13 February 2011).

Descripción de la metodología paso a paso:

  1. A strain of bacteria, Planococcus sp., with a diameter < 1 μm was labeled with BrdU (5′-bromo-2′-deoxyuridine, Sigma B5002) by inoculating into 50 mL of fresh media (1% yeast extract, 0.22 μm filtered seawater) with 20 μM BrdU. Planococcus for control incubations was treated identically except that BrdU was excluded from the media. The bacterial cells were harvested by centrifugation (3,000 × g, 10 min), washed 3 times, and dispersed and resuspended by pipetting with cold phosphate-buffered saline.
  2. There were two treatments per experiment; the addition of BrdU-labeled Planococcus and the addition of unlabeled Planococcus. Planococcus was added at 10% of the natural abundance of bacteria for the first experiment and at 20% for the remaining three BrdU incubation experiments. All cubitainers were incubated at −0.5°C in continuous fluorescent light at ~7 × 1015 quanta s−1 cm−2 (surface samples) and ~7 × 1014 quanta s−1 cm−2 (CM samples). Samples (1–2 L) were taken after 72 h of incubation for each depth/replicate, collected on 0.2 μm Durapore filters and frozen at −80°C for later extraction.
  3. DNA was extracted following the protocol in Gast et al. (2004), resuspended in 100 μL 1 × Tris/EDTA and stored frozen at −80°C for transport. Immunoprecipitation of BrdU-labeled DNA was performed following the protocol modified and published by Fay et al. (2013). Samples of DNA from incubations that were fed unlabeled bacteria are identified as “Whole,” whereas the samples fed BrdU-labeled bacteria are identified as “IP” (immunoprecipitated).
  4. Primers to amplify the V9 region of the 18S ribosomal RNA gene were used to generate products for amplicon high throughput sequencing. For each sample, 3 PCR reactions were performed at a volume of 50 μl each that contained 0.2 μM of each primer, 1× reaction buffer, 200 μM each dNTP, and 0.5U Phusion DNA Polymerase (New England Biolabs F-553). Cycling conditions were: 98°C for 120 s; 10 cycles of 98°C for 15 s, 67°C decreased by 1°C cycle−1 for 20 s, and 72°C for 15 s; 25 cycles of 98°C for 15 s, 57°C for 20 s, and 72°C for 15 s; and a final extension step of 72°C for 120 s. Each sample was purified with AMPure XP beads (Beckman Coulter A63880) following the manufacturer's protocol.
  5. Sequencing was accomplished on pooled triplicate samples at the University of Pennsylvania DNA Sequencing Facility using a “1-way read” approach for amplicon pyrosequencing with the GS Junior emPCR Lib-L kit (454 Life Sciences) on a Roche/454 GS-FLX.

Referencias Bibliográficas

  1. Gast, R. J., Fay, S. A., & Sanders, R. W. (2018). Mixotrophic Activity and Diversity of Antarctic Marine Protists in Austral Summer. Frontiers in Marine Science, 5, 13.

Metadatos Adicionales

Identificadores Alternativos b9c72483-b00a-4848-9239-b36dce06b0cb
https://ipt.biodiversity.aq/resource?r=marine_microbial_eukaryotes_18s_experiment_rosssea