Metagenomes of oyster gut microbiomes in Narragansett Bay RI
Total nucleic acids (TNA) were extracted from 150-200 mg of gut tissue (n=10 oysters per site; 50 total) using the Qiagen Allprep PowerViral DNA/RNA extraction kit with modifications as follows. The gut tissue sample was added directly to a 0.1 mm glass bead tube (Qiagen), along with 600 uL of Solution PV1 and 6 uL of sterile beta-mercaptoethanol to minimize RNA degradation. The samples were subjected to bead beating for 5 minutes, followed by proteinase K digestion at 55 C for 1 hour in a shaker at 300 rpm. The supernatant was transferred to a new microcentrifuge tube and the protocol continued according to the manufacturers recommendations. Following TNA extraction, the concentration was quantified using a Nanodrop 2000 instrument (ThermoFisher), and 30 uL was allocated for DNA. DNA purification of the 30 uL TNA aliquot was performed using an adapted version of the DNeasy PowerLyzer PowerSoil Kit. In brief, the TNA aliquot was transferred to a new 2 mL microcentrifuge tube and 1200 uL of Solution C4 was added, then vortexed to mix. Next, 4 uL of RNase A solution was added to the sample and incubated for 2 minutes at room temperature. The treated DNA was then purified using the spin column and an ethanol wash with Solution C5. The final DNA sample was eluted in 50 uL of Solution C6. The metagenomic libraries were prepared from 4?g of gut DNA (n=5 per site) and fragmented at 500nt using Covaris ultrasonification prior to library prep. Illumina TruSeq PCR-free library kits were used to prepare the libraries, and then verified using both a KAPA library quantification kit and an Agilent Bioanalyzer. The resulting metagenomic libraries were sequenced on an Illumina NovaSeq S4 to obtain 2x150bp paired-end reads at the Yale Center for Genome Analysis.