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

Последняя версия опубликована SCAR - Microbial Antarctic Resource System 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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Исследователи должны дать ссылку на эту работу следующим образом:

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

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Публикующей организацией и владельцем прав на данную работу является SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 License.

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Этот ресурс был зарегистрирован в GBIF, ему был присвоен следующий UUID: b9c72483-b00a-4848-9239-b36dce06b0cb.  SCAR - Microbial Antarctic Resource System отвечает за публикацию этого ресурса, и зарегистрирован в GBIF как издатель данных при оподдержке Scientific Committee on Antarctic Research.

Ключевые слова

Metadata

Контакты

Кто является создателем ресурса:

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

Кто может ответить на вопросы о ресурсе:

Rebecca Gast
Woods Hole Oceanographic Institution Woods Hole US

Кем заполнены метаданные:

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

Кто еще связан с данным ресурсом:

User

Географический охват

Southern Ocean: Ross Sea, Antarctica

Ограничивающие координаты Юг Запад [-75.44, -171.29], Север Восток [-72.35, 178.3]

Таксономический охват

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

Domain  Eukaryote (Eukaryotes)

Временной охват

Дата начала / Дата окончания 2011-01-26 / 2011-02-08

Данные проекта

Описание отсутсвует

Название National Science Foundation Grants OPP-0838955 (RG) and OPP-0838847 (RS)
Финансирование This work was supported by National Science Foundation Grants OPP-0838955 (RG) and OPP-0838847 (RS).

Исполнители проекта:

Rebecca Gast

Методы сбора

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.

Охват исследования 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).

Описание этапа методики:

  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.

Библиографические ссылки

  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.

Дополнительные метаданные

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