Total RNA was extracted from RNAlater®-preserved samples using the Ambion RNAqueous®-Micro Total RNA Isolation kit. First-strand cDNA was constructed using the SMART® cDNA Library Construction Kit (Clontech Laboratories, Inc.), replacing the included 3′ primer with the Cap-TRSA-CV oligo [19 (link)]. We amplified double-stranded cDNA using the Advantage® 2 PCR Kit (Clontech Laboratories, Inc.). To minimize the risk of contamination, extractions and cDNA construction were performed in small batches of four tissue samples or fewer, and the workstation and tools were cleaned with bleach between each set of extractions. Where possible, we avoided sampling the external body surface and the gut to limit the potential for contamination from epibionts and gut contents (e.g., prey items and microorganisms).
Non-normalized cDNA libraries were sent to Hudson Alpha Institute for Biotechnology, Huntsville, Alabama USA for library preparation and 2 × 100–bp paired-end sequencing on an Illumina HiSeq 2000. Approximately one-sixth of a lane was used for each taxon.
Non-normalized cDNA libraries were sent to Hudson Alpha Institute for Biotechnology, Huntsville, Alabama USA for library preparation and 2 × 100–bp paired-end sequencing on an Illumina HiSeq 2000. Approximately one-sixth of a lane was used for each taxon.
Free full text: Click here
