RNA-Seq of Diadema antillarum: coelomocytes
Six live adult sea urchins were collected in southwestern Puerto Rico. The animals were kept in a 75-gallon marine aquarium for three months at the Department of Marine Sciences at UPRM on Magueyes island. The extended housing was to establish near immune quiescent animals or otherwise promote animals with down-regulated immune systems (Gross et al. 2000; Clow et al. 2000). Following the three months of housing, animals (n = 3) received two separate  injections of lipopolysaccharide (LPS; 1.0 g/l; Sigma-Aldrich Co. St. Louis MO) in artificial coelomic fluid (aCF; see recipe in Brockton et al. 2008).  The injection was administered through the peristomal membrane using a sterile 23-gauge needle connected to a 1-mL syringe. Each animal (SeaUrchin1_3) received 1 g LPS per ml of coelomic fluid (CF), which is known to elicit an immune response (Smith et al. 1995; Terwilliger et al. 2007). The total volume of CF per animal was calculated according to Smith et al. 1995. The other animals (n = 3) received a sham injection of aCF (SeaUrchin4_6). The initial injection was administered at 0 hr followed by a second injection 24 hr later. A sample of wCF was collected from each animal 24 hr after the second injection.  Coelomocytes were pelleted in 1.5 ml tubes at maximum speed (21, 300 x g) for 2 min at room temperature in a benchtop Eppendorf 5425 centrifuge. The cell free fluid was removed and replaced with RNAlater solution. Samples were stored in a -20 C freezer prior to RNA extraction. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) by following the manufacturers instructions. The sample quality and concentration were assessed using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific).  Samples were pooled together on an equimolar basis for LPS injected or aCF injected animals.  The two samples were shipped on dry ice for sequencing at the Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD 20847, USA. To prepare the samples for sequencing, ribosomal RNAs were removed using a RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen). Libraries were prepared using a Total RNA-Seq Kit v2 (Life Technologies). Quality and quantitation verification of libraries was performed using a Bioanalyzer 2100. Beads were prepared using the Ion PI Template OT2 200 Kit v2 on a One Touch 2 System (Life Technologies). Next, samples were prepared for sequencing using an Ion Proton 200 sequencing kit v2 (Life Technologies). Each sample was sequenced on a PI Ion Chip in an Ion Proton Sequencer.