title: Metatranscriptomes of oyster microbiomes with nutrient enrichment
design: Total nucleic acids (TNA) were extracted from 150-200 mg of gut tissue 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 ?L of Solution PV1 and 6 ?L of sterile ?-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 ?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. The metatranscriptomic libraries were prepared from 300ng of RNA and fragmented at ~400nt. rRNA depletion and library prep were performed using the Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research Cat. No. R3000), and verified using TapeStation D5000. The resulting metatranscriptomic libraries were sequenced on an Illumina HiSeq 2500 to obtain 2x100bp paired-end reads at Zymo Research.