0 (Bruker Daltonics, Billerica, USA). Software GPMAW 9.0 (Lighthouse Data, Denmark) [66] was used for the theoretical calculations of molecular masses. Sea anemone peptide sequences used for calculation of molecular masses of known toxins were extracted from [63]. Venom maps were constructed by using Microsoft Excel 2007 (Microsoft, USA). The histograms

were constructed with the statistical software Origin 6.0 (Microcal Software, MA, USA). Based on the results obtained in the molecular check details masses measurements of the peptides, as well as the higher abundance of toxins in B. granulifera, we decided to focus our analysis only on the transcriptomics of this species. The total RNA was extracted from tentacles tissues of B. granulifera specimens using the TRIZOL® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. DNA digestion was performed using DNase I (Sigma, St. Louis, MO). After DNase I treatment, total RNA was purified using RNeasy Mini Spin Column (Qiagen). The quality and quantity of the total RNA were detected using RNA 6000 pico LabChip® kit in Agilent 2100 bioanalyzer. Also, the total RNA quantity learn more was measured using Quant-iT™ RNA

BR Assay Kit in Qubit® 2.0 Fluorometer. cDNA was synthesized using Roche double-stranded cDNA synthesis kit (Roche Applied Sciences, USA) from total RNA with oligo (dT) 20 primer. A cDNA library was prepared using cDNA rapid library preparation method kit (Roche Applied Science, GS Junior Titanium Series, USA) according to manufacturer’s instruction. Approximately 500 ng of DNA was fractionated into smaller fragments (300–500 4��8C base pairs) that are subsequently polished (blunted) and subjected to adaptor ligation. The optimal amount of cDNA was adjusted to single DNA copy per bead for emulsion PCR (emPCR). Finally, the sequencing was performed in the GS Junior pyrosequencing system (Roche 454 Life Sciences, Branford, CT, USA). EST reads were assembled to contigs by using GS Junior Assembler software. Contigs were mapped to the NCBI non-redundant

databases using MAQ (v0.7.1) [49]. The following softwares were used in the present work: FASTA, http://www.ebi.ac.uk/Tools/sss/fasta/[65] for identifying related sequences retrieved from UniProt Knowledgebase; Clustalw2.1, http://www.ebi.ac.uk/Tools/clustalw2/index.html[48] for multiple sequence alignment of new peptides and related sequences; Jalview, http://www.jalview.org/[83] for illustrating conserved amino acid; I-TASSER, http://zhanglab.ccmb.med.umich.edu/I-TASSER/[70] and [87] for peptide molecular modeling and structure prediction; Deep View Swiss-pdb Viewer 4.0.1, http://www.expasy.org/spdbv/[33] for viewing and calculation of electrostactic potentials of the peptide structures and models and PyMol (The PyMOL Molecular Graphics System, Version 1.2, 254 Schrödinger, LLC., http://www.pymol.org/) for viewing the structures and models.