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

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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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此資料的發布者及權利單位為 SCAR - Microbial Antarctic Resource System。 This work is licensed under a Creative Commons Attribution (CC-BY 4.0) License.

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此資源已向GBIF註冊，並指定以下之GBIF UUID: b9c72483-b00a-4848-9239-b36dce06b0cb。  SCAR - Microbial Antarctic Resource System 發佈此資源，並經由Scientific Committee on Antarctic Research同意向GBIF註冊成為資料發佈者。

關鍵字

Metadata

聯絡資訊

地理涵蓋範圍

Southern Ocean: Ross Sea, Antarctica

分類群涵蓋範圍

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

時間涵蓋範圍

計畫資料

無相關描述

參與計畫的人員:

取樣方法

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.

方法步驟描述:

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.

引用文獻

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.

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