Metatranscriptomes of oyster microbiomes with nutrient enrichment
Total nucleic acids (TNA) were extracted from the swab samples using the Qiagen Allprep PowerViral DNA/RNA extraction kit with modifications as follows. The swab samples were removed from the RNAlater and each washed with 1 mL of sterile, filtered 1X nuclease-free Phosphate Buffer Saline (PBS, pH 7.4, Invitrogen). The remaining RNAlater was centrifuged to pellet the excess cells, then washed with 1X PBS. The cell pellet was suspended in 600 ?L of Qiagen Solution PV1 and added directly to a 0.1mm glass bead tube, along with the washed swab and 6 ?L of sterile ?-mercaptoethanol. 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 ?L was allocated for RNA. RNA purification from the 30 ?L TNA aliquot was performed using the DNase Max I kit according to the manufacturers protocol in a 50 ?L reaction volume. RNA from 3 oyster samples per bucket were pooled equimolarly for the metatranscriptomes. The metatranscriptomic libraries were prepared from 2?g of pooled RNA, fragmented at 500nt using Covaris ultrasonification, and treated with the Illumina Ribo-Zero Gold rRNA Removal Epidemiology Kit prior to library prep to remove both host and bacterial rRNA. 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 metatranscriptomic libraries were sequenced on an Illumina NovaSeq S4 to obtain 2x150bp paired-end reads at the Yale Center for Genome Analysis.