16S amplicon of oyster microbiomes with nutrient enrichment
Total DNA from water samples was extracted from the Sterivex filters using the Qiagen Allprep PowerViral DNA/RNA and DNeasy PowerLyzer PowerSoil kits with modifications as follows. The RNAlater was flushed out of the filters using a sterile syringe, then the filters were rinsed with 2 mL of 1X sterile nuclease-free Phosphate Buffer Saline (PBS, pH 7.4, Invitrogen). Solution PV1 (1800 ?L) and 18 ?L of sterile ?-mercaptoethanol were added directly to the filter cartridge and incubated at 37 C for 30 minutes. Next, 20 ?L of proteinase K was added to the filter and digested at 55C for 1 hour. The supernatant was flushed from the filter, transferred to 3 new microcentrifuge tubes and the protocol continued according to the manufacturers recommendations. DNA was purified from the entire TNA product using the methods described above. Following extraction, DNA concentrations were quantified with both a Nanodrop 2000 instrument (ThermoFisher) and Qubit Fluorometer High-Sensitivity reagents (Invitrogen). 16S rRNA gene amplicon analysis was performed using 967F/1064R primers to amplify the V6 region.