CV_before
aquarium metagenome
CV_before
isolation_source
zebrafish aquarium water
collection_date
2024-11
geo_loc_name
USA: Durham
lat_lon
not applicable
samp_collect_device
Before conventionalization of germ-free embryos at 6 hpf, 1 mL of recirculating system water was flash frozen and stored at -80C until further processing. At 32 hpf, at endpoint collection, 1 mL of water from each of the conventionalized flasks (CV6 - conventionalized at 6 hpf, and CV24 - conventionalized at 24 hpf) flasks were flash frozen and stored at -80C until further processing. Experiments were repeated three times.
samp_mat_process
DNA was extracted by The Duke Microbiome Core Facility using a PowerSoil Pro kit (QIAGEN, 47014) with the following modifications. 500 L of water sample was added to PowerWater DNA Bead Tubes (QIAGEN, 14900-50-NF-BT) with 1.6 mL of extraction buffer from the PowerSoil Pro kit. The mixture was incubated at 65C for 10 minutes and vortexed at maximum speed for 5 minutes on Vortex-Genie 2 with 5 mL adapters. The manufacturer's instructions for PowerSoil Pro kit (QIAGEN, 47014) were then followed with buffer volumes adjusted to account for higher starting volume. Bacterial community composition in isolated DNA samples was characterized by amplification of the V4 hypervariable region of the 16S rRNA gene by polymerase chain reaction (PCR) using the forward primer 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') from the Earth Microbiome Project protocol (http://www.earthmicrobiome.org/). The reverse primers (806R) carry unique barcodes that allow for multiplexed sequencing. PCR was performed using Phusion Plus PCR Master Mix (ThermoScientific F631L) for a total of 40 cycles. PCR products were purified with AMPure XP Beads (Beckman Coulter A63881), and concentrations were assessed using a Qubit dsDNA HS assay kit (ThermoFisher, Q32854) and a Promega GloMax plate reader. Equimolar 16S rRNA PCR products from all samples were pooled prior to sequencing. Sequencing was performed by the Duke Sequencing and Genomic Technologies shared resource on an Illumina MiSeq instrument configured for 250 base-pair paired-end sequencing runs.
samp_size
1 mL
Sample ID
CV_before_02
BioSampleModel
Metagenome or environmental