alias: 6_Gut_6
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. 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.
identifiers.namespace: SUB6800262
identifiers.id: 6_Gut_6