Methodology described in Drake et al. 2013:
SOM Extraction: S. pistillata skeletons were soaked for 4 hours in 3% (wt/vol) sodium hypochlorite, copiously rinsed in deionized water, and dried overnight at 60 degrees C. Dried skeletons were ground to a fine powder with an agate mortar and pestle and again bleached, rinsed, and dried. The skeletal powder was decalcified in 1 N HCl at room temperature while shaking. HCl was added gradually so that the solution reached neutral pH within 30 min of acid addition; more HCl was only added if skeleton powder remained after 30 min. pH of the decalcification solution was brought to neutral with 1 M NaOH. Water-soluble and -insoluble organic fractions were separated by centrifugation and analyzed separately. Trichloroacetic acid (TCA)-acetone precipitations were used to clean and precipitate proteins from the decalcification solution. Briefly, one volume of 60% (wt/vol) TCA was added to five volumes soluble SOM samples and 1 mL 60% (wt/vol) TCA was added to insoluble SOM pellets. Both fractions were incubated at 4 degrees C overnight, centrifuged at 10,000 x g at 4 degrees C for 30 min, washed twice with ice-cold 90% (vol/vol) acetone at 4 degrees C for 15 min, and centrifuged at 10,000 x g at 4 degrees C for 30 min. Additionally, SOM proteins were enzymatically deglycosylated with O-glycosidase, N-glycosidase F, sialidase, B1-4 galactosidase, and B-N-acetylglucosaminidase in a deglycosylation mix per manufacturer instructions (New England BioLabs).

Protein Separation and Characterization: SOM proteins were separated by SDS/PAGE and bands were visualized by silver staining (Pierce silver stain for mass spectrometry) and Periodic acid-Schiff staining (Pierce glycoprotein staining kit). Smearing of proteins in gels precluded extraction of individual bands for sequencing.

Proteomics: SOM complexes were digested either by trypsin or proteinase K, and masses and charges of the digested peptides were analyzed on a Thermo LTQ-Orbitrap-Velos ETD mass spectrometer with Dionex U-3000 Rapid Separation nano LC system. The LC-MS/MS data were searched using predicted gene models from S. pistillata by X! Tandem using an in-house version of the Global Proteome Machine (GPM USB; Beavis Informatics) with carbamidoethyl on cysteine as a fixed modification and oxidation of methionine and tryptophan as a variable modification. Spectra were also analyzed against a suite of potential microbial genomes to exclude possible microbial contamination of the dry skeleton. Data for LC-MS/MS sequenced proteins have been deposited in GenBank.

Gene Confirmation: Internal sequences of predicted genes were confirmed in DNA and cDNA by PCR using gene-specific primers (Table S2). Holobiont DNA and cDNA were prepared as previously described from S. pistillata colonies maintained in in-house aquaria. All PCR tubes contained 0.25-ug template, 0.2 mM dNTPs, 1 x High Fidelity reaction buffer, 0.5 uM of each primer, and 0.04 units uL−1 of Phusion polymerase (New England BioLabs) in a 25-uL reaction volume. Amplifications were performed in a Veriti Thermal Cycler (Applied Biosystems) at 35 cycles of 98 degrees C for 10 s, primer-specific annealing temperature for 30 s, and 72 degrees C for 30–180 s. PCR products were sequenced by GENEWIZ.

Bioinformatics: LC-MS/MS results were filtered to remove hits from standard contamination (common Repository of Adventitious Proteins, or cRAP, database). A nonredundant list of all proteins detected with e-values ≤10−10 was used for blast analysis against NCBI and to query a database we created that contains translated sequences from Homo sapiens, Thalassiosira pseudonana [diatom], Nematostella vectensis [anemone], Strongylocentrotus purpuratus [urchin], E. huxleyi CCMP1516 (coccolithophore; draft genome), and A. digitifera [hard coral] genomes; a transcriptome from P. damicornis [hard coral]; and expressed sequence tag (EST) libraries from Favia sp. [hard coral], Reticulomyxa filosa [foraminiferan], and P. maxima [oyster]. N. vectensis and R. filosa do not biomineralize; all other comparison species produce calcium- or silica-based minerals. Predicted proteins from the comparison species with similarities greater than 35% and e-values ≤10−10 were retained for further analysis. For CARP subfamily homologs, predicted proteins in comparison species were combined if they closely mimicked matched S. pistillata CARPs. These combinations are noted in protein names when they are presented in the multiple sequence alignment. Residues whose conservation suggests a functional role were predicted in ConSurf using CARP4 as the query sequence. Structures of selected proteins were predicted using both I-TASSER and Phyre2. We used these two programs to obtain a consensus in structure matching, particularly in the case of one S. pistillata protein that showed no similarity to proteins in the NCBI and contained no known domains. Glycosylation sites were predicted using the EnsembleGly server at the AIRL at Iowa State University. Images of predicted structures were generated in MacPyMOL v1.3r1 (Schrodinger).