A clonal line of Aiptasia pallida (clone CC7, available through the Pringle lab) hosting Symbiodinium of clade A was established from a single tiny propagule in a population obtained from Carolina Biological Supply (Burlington, NC) and grown into an abundant stock. Given the Symbiodinium clade harbored by this population, it is likely that the Aiptasia individual originated from the Florida Keys lineage. Approximately 500 anemones of various sizes were harvested from this stock under normal growth conditions (~26°C; salinity, ~33 ppt; light, ~40 μmol m-2 s-1 photosynthetic photon flux; 12-h light-dark cycle), blotted to remove excess water, and immediately frozen in liquid nitrogen. The anemones were then ground to a fine powder under liquid nitrogen using a ceramic mortar and pestle. The powder was weighed (~4 g) while still frozen and mixed with a proportional volume (50 ml) of TRIzol Reagent (Invitrogen, Carlsbad, CA); extraction was then performed in accordance with the manufacturer's instructions yielding ~5 mg of total RNA. This RNA was sent to Open Biosystems (Huntsville, AL), where it was tested for quality; mRNA was then isolated using oligo(dT)-coated magnetic particles (Seradyn, Indianapolis, IN), and cDNA was synthesized. Double-stranded cDNA was size fractionated to enrich for long reads, cloned into the vector pExpress1 (Express Genomics, Frederick, MD), and electroporated into E. coli strain DH10B. The resulting library was determined to contain ~96% recombinants with an average insert size of 1.95 kb. Sequencing was performed on 96-well capillary sequencing platforms (ABI 3700) at the DOE Joint Genome Institute (JGI, Walnut Creek, CA) and at the Genome Core Facility at the University of California, Merced, USA, CA.
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