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Palmer LTER 2014 cruise microbial Eukaryote (18s) plankton
Citatie
Lin Y, Cassar N, Marchetti A, Moreno C, Ducklow H, Li Z (2018): Palmer LTER 2014 cruise microbial Eukaryote (18s) plankton. v1.3. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=palmer_lter_eukaryote_18s_plankton&v=1.3 https://doi.org/10.15468/wtt5gg
Contact: Yajuan, Lin

Toegang tot data
Gearchiveerde data
Beschikbaarheid: Creative Commons License Deze dataset valt onder een Creative Commons Naamsvermelding 4.0 Internationaal-licentie.

Beschrijving
Amplicon sequencing dataset of microbial eukaryotes (18S ssh rRNA v4)living in the plankton of the Southern Ocean around the Western Antarctic Peninsula. Samples were collected during the annual Palmer LTER cruise from Jan to early Feb in 2014 aboard the R/V Laurence M. Gould. meer

Surface seawater from the vessel seawater supply line was gently vacuum filtered through a 0.2 µm pore size Millipore Supor filters. Each filter was then preserved immediately in 1 ml of RNA-later and stored at −80 °C.
Samples were taken during the annual Palmer LTER cruise from Jan to early Feb in 2014 aboard the R/V Laurence M. Gould.
Method step description:
  1. From each sampled station one filter was first split in two halves, one for DNA extraction and the other for later RNA studies. 500 µl of the RNAlater from the original tube was loaded onto an Amicon 10 k column and the column was spun for 15 minutes at 14,000 g to get rid of most of the RNAlater and concentrate cells suspended in RNAlater. The resulting solution (~50 µl) was then added back to the one half filter. The cells on filter were lysed by bead-beating using 0.2 g of zirconium beads in 400 µl of buffer AP1 (Qiagen).
  2. DNA was extracted and purified using Qiagen DNeasy Plant Mini kit following the manufacture’s instruction. 18 S (eukaryotic) rDNA gene fragments were amplified by PCR using V4 primer sets: 18 S forward (5′- CCAGCASCYGCGGTAATTCC-3′) and reverse (5′-ACTTTCGTTCTTGAT-3′). Amplicons from each sample were tagged using dual index fusion primers and a “heterogeneity spacer” was used to improve sequencing quality. The “heterogeneity spacer” is a 0–5 bp of spacer added to the index sequence in order to allow different samples be sequenced out of phase and thus improve the amplicon library sequencing quality. Each PCR reaction (25 µl) consisted of 1U of Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 1 × High Fidelity Buffer, 200 µM dNTPs, 2 mM MgSO4, 0.2 µM each primer, and 5–30 ng extracted environmental DNA template. PCR was conducted with an initial activation step at 94 °C for 3 min, followed by 30 three-step cycles consisting of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min, and a final extension step of 72 °C at 10 min. PCR products in triplicates for each sample were then pooled and purified using QIAquick PCR Purification kit, and the concentration of amplicon DNA was quantified using a Qubit dsDNA assay.
  3. Amplicons from different locations were pooled in equimolar amounts (final concentration ~10 ng/µl) and submitted to Duke Institute for Genomic Sciences and Policy (IGSP) for sequencing using Illumina MiSeq. 300PE platform.

Scope
Thema's:
Biologie > Plankton
Kernwoorden:
Marien/Kust, Eukaryotes, Metadata, PS, Zuidelijke Oceaan

Geografische spreiding
PS, Zuidelijke Oceaan [Marine Regions]

Spreiding in de tijd
Januari 2014 - Februari 2014

Parameter
Moleculaire data

Bijdrage door
Duke University, meerdata creator

Gerelateerde datasets
Gepubliceerd in:
AntOBIS: Antarctic Ocean Biodiversity Information System, meer
(Gedeeltelijk) opgenomen in:
RAS: Register of Antarctic Species, meer

Dataset status: Afgelopen
Data type: Meta database
Data oorsprong: Onderzoek: veldonderzoek
Metadatarecord aangemaakt: 2018-12-12
Informatie laatst gewijzigd: 2019-04-10
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