The effects of cold temperature on the development, microbiome, and transcriptome of the sea anemone Nematostella vectensis
Adult anemones (n = 12) were held at room temperature and starved 48 hours prior to initiating the experiment. Prior to thermal exposures, 12 anemones were preserved in ethanol and placed at -80C. Then single anemones were placed in a 12-well plate with filter-sterilized seawater and placed in appropriate temperatures, 0C, 10C, 20C. Twenty-four hours and seven days later 12 anemones were immediately placed into molecular grade ethanol and stored in -80C until extraction. Total DNA was extracted using the QIAgen DNeasy extraction kit. The 16S V1-V2 variable gene region was amplified using the primers TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CCTACGGGNGGCWGCAG and GTCTCGTGGGCTCGGAGATGTGTATAAGAGAC AGGACTACHVGGGTATCTAATCC (Eurofins). The Nextera Set A barcode kit was used in a secondary PCR to add barcodes. The resulting amplicons were pooled and sequenced on the Illumina NextSeq 2x300bp. Following paired end sequencing, the reads were analyzed using QIIME2. We rarified the data to 10,190 reads, which retained 11 of 12 samples for all conditions. The taxonomies of the amplicon sequence variants (ASVs) were assigned with a Naive Bayes classifier with the SILVA database trained for 16S V1/V2 primers used to amplify the region.