Collection

Specimens of Diadema antillarum (urn:lsid:marinespecies.org:taxname:124332) were collected by snorkel and transported to the lab in seawater buckets. The specimens were photographed, and vivisected into tissue components (body wall, spine, gonad, digestive tract and coelomic fluid), which were then frozen in liquid N2 for transport to the laboratory at Cornell University.

Transcriptomic Sequencing

Transcriptomes from coelomic fluid of grossly normal (n = 2) and abnormal (n = 2) individuals from an affected site and of grossly normal individuals from a reference site (n = 2) were prepared alongside grossly normal (n = 2) aboral body wall tissues from a reference site to examine expressed genes and microorganisms associated with infection. RNA was extracted from coelomic fluid (300 μl) and body wall tissues (200 mg) using the Zymo Quick-RNA Tissue/Insect Kit using on-column deoxyribonuclease I digestion per the manufacturer’s protocols. Transcriptome libraries were prepared from purified RNA extracts using the Zymo RiboFree kit and sequenced on a single run of Illumina MiSeq using a Nextera DNA Library Prep Kit at the Biotechnology Resource Center at Cornell University. All sequences have been deposited at National Center for Biotechnology Information (NCBI) Sequence Read Archive under Bioproject PRJNA900371 and SRA accession numbers SRR22260770 to SRR22260777.

Viral Metagenomic Sequencing

Small subsamples of each specimen were prepared for viral metagenomic sequencing broadly following the approach of Ng et al. (2011). Briefly, tissues were homogenized in sterile, 0.02 µm filtered phosphate buffered saline by beating using Zymo ZR Basher Beads (part number S6012-50) in a Biospec Instruments homogenizer for 1 min at maximum speed. The homogenates were centrifuged at 3,000 × g for 1 min to remove large cell debris, before filtering the supernatant through 0.2 µm pore size polyethersulfone syringe filters. Filtered homogenates were treated with 5U Turbo DNase (Invitrogen; part number AM2238), 5U Benzonase nuclease (Sigma Aldrich; part number E1014) and 20U RNase ONE (Promega; part number M4261) for 2 hours at 37 °C to digest extracellular and non-ribosome-bound nucleic acids. Following incubation, RNA was extracted from subsamples of purified virus-sized material using the Zymo Viral RNA kit (part number R1034) before storing RNA at −80 °C until further processing.

RNA in viral extracts was amplified using the SeqPlex RNA kit (Sigma Aldrich; part number SEQXE-10RXN, n = 11 libraries). Subsequent specimens were amplified using the TransPlex kit (WTA2; Sigma Aldrich; part number WTA2-10RXN, n = 3 libraries), which was used in prior RNA viral metagenomic work. Amplification products were purified using the Zymo DNA Clean and Concentrator -5 (part number D4004), quantified by Pico Green fluorescence (Invitrogen; part number P11496), and submitted for sequencing at the Cornell Biotechnology Resource Center. Libraries were prepared for sequencing using the Nextera Flex kit (Illumina; part number 20018704) and sequenced on the Illumina MiSeq platform using the 500 bp Nano kit. Sequences from all libraries were archived at NCBI under Bioproject PRJNA1117494 and SRA accessions SRR29258987 to SRR29258999.