title: PAC3
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-06-30
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-06-30
attributes.tag: External Id
attributes.value: SAMEA7129494
attributes.tag: INSDC center alias
attributes.value: DEPARTMENT OF ZOOLOGY
attributes.tag: INSDC center name
attributes.value: DEPARTMENT OF ZOOLOGY
attributes.tag: INSDC first public
attributes.value: 2022-06-30T00:17:43Z
attributes.tag: INSDC last update
attributes.value: 2022-06-30T00:17:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: PAC3
attributes.tag: collection date
attributes.value: 2016-08-17
attributes.tag: environment (biome)
attributes.value: coral reef
attributes.tag: environment (feature)
attributes.value: marine biome
attributes.tag: environment (material)
attributes.value: water
attributes.tag: geographic location (country and/or sea)
attributes.value: Virgin Islands
attributes.tag: geographic location (depth)
attributes.value: 7
attributes.tag: geographic location (latitude)
attributes.value: 18.312023
attributes.tag: geographic location (longitude)
attributes.value: -64.723135
attributes.tag: geographic location (region and locality)
attributes.value: Tektite Cliffs, St. John, USVI
attributes.tag: host common name
attributes.value: Ramicrusta textilis
attributes.tag: host taxid
attributes.value: 680470
attributes.tag: investigation type
attributes.value: bacteria_archaea
attributes.tag: nucleic acid amplification
attributes.value: For analysis of the microbiome, the V4 region of the small-subunit (SSU) 16S rRNA gene was amplified from genomic DNA using a two-step nested PCR approach. Briefly, samples were amplified using primers 519F and 806R. The reaction mixture consisted of 10 μL HotStarTaq Master Mix (Qiagen), 500 nM of each primer, 1 μL template DNA, 200 ng mL-1 non-acetylated BSA and nuclease-free water to bring the total volume to 20 μL. In the second step, pooled amplicons were amplified and barcoded by nested PCR using MID-tagged primers 519F and 806R in a reaction mixture comprising 25 μL HotStarTaq Master Mix, 500 nM of each primer, 1 μL amplicon DNA, and nuclease-free water to bring the total volume to 50 μL.
attributes.tag: nucleic acid extraction
attributes.value: Genomic DNA was extracted from mucous swabs using a modified protocol for the Fast DNA SPIN Kit for Soil (MP Biomedicals). Briefly, the contents of the Lysing Matrix E tube were transferred to the sample vial containing the cotton swab. Sodium Phosphate Buffer (300 μL) was added to the sample and vortexed for 5 s. MT Buffer (122 μL) was added to the cryogenic vial and the sample homogenised on a Pulsing Vortex Mixer (VWR) at maximum speed for 40 s. The sample was centrifuged at 10,000 x g for 10 min and the supernatant transferred to a 2 mL tube. Solution PPS (250 μL) was added to the supernatant and mixed by inversion ten times. The sample was centrifuged again at 14,000 x g for 5 min and the supernatant transferred to 15 mL tube. The Binding Matrix suspension was resuspended by vortexing for 5 s and 1 mL immediately added to the supernatant, before mixing by inversion for 2 min and settling in a rack for 3 min. Avoiding the Binding Matrix, 500 μL supernatant was removed and discarded and the remaining supernatant and Binding Matrix resuspended by inversion, before 600 μL suspension was transferred to a SPIN filter. The SPIN filter was centrifuged at 14,000 x g for 1 min and the filtrate discarded. The remaining suspension was added to the SPIN filter and centrifuged again at 14,000 x g for 1 min. The filtrate was again discarded and 500 μL SEWS-M added to the pellet on the SPIN filter and the pellet gently resuspended using the pipette. The SPIN filter was centrifuged at 14,000 x g for 1 min, the filtrate discarded and the SPIN filter centrifuged again at 14,000 x g for 2 min. The catch tube containing the filtrate was discarded and the SPIN filter placed in a clean catch tube and air-dried for 5 min. The pellet on the SPIN filter was gently resuspended in ddH2O (100 μL) using a pipette and the SPIN filter incubated for 5 min at 55°C, before a final centrifugation at 14,000 x g for 1 min. The SPIN filter was discarded and the eluate stored at -20°C.
attributes.tag: pcr conditions
attributes.value: PCR1 (25 cycles): initial denaturation=95degC_20s; annealing=55degC_30s; extension=72degC_30s; final extension=72degC_7min; PCR2 (15 cycles): initial denaturation=95degC_20s; annealing=62degC_30s; extension=72degC_30s; final extension=72degC_7min
attributes.tag: pcr primers
attributes.value: 521F (5'-CAG CMG CCG CGG TAA-3'), 806R (5'-GGA CTA CHV GGG TWT CTA AT-3')
attributes.tag: plant-associated environmental package
attributes.value: plant-associated
attributes.tag: project name
attributes.value: Characterisation of the Microbiome of a spatially-aggressive Peyssonnelid Algal Crust (PAC)
attributes.tag: sample collection device or method
attributes.value: Cotton swab
attributes.tag: sample material processing
attributes.value: Sterile cotton swabs were stored prior to sampling in sealed, sterile 15 mL polypropylene tubes. Inverted tubes were opened underwater immediately above the crustose algae and their SM was collected by rolling the swab over the algal surface, and returning the swabs to the inverted tube such that it was sealed with minimal seawater ingress and the swab maintained in an air pocket. Upon return to the laboratory (<60 min after sampling), swabs were trimmed to 2 cm length and added to cryogenic vials (2 mL; Corning, Part No. 430488) containing 500 μL RNAlater (ThermoFisher Scientific), with the swab fully immersed. Cryogenic vials were stored at -20°C in St. John for five days, but were thawed to room temperature for shipment to Europe where they were stored at 4°C prior to analysis.
attributes.tag: sample name
attributes.value: PAC3
attributes.tag: sample storage duration
attributes.value: 14
attributes.tag: sample storage temperature
attributes.value: 4
attributes.tag: sample volume or weight for DNA extraction
attributes.value: 1
attributes.tag: sequencing method
attributes.value: Illumina MiSeq
attributes.tag: target gene
attributes.value: 16S
attributes.tag: target subfragment
attributes.value: V4
attributes.tag: temperature
attributes.value: 30