Besides, the evolutionary origin of the cirripede crustacean lineage remains ambiguous ( Meusemann et al., 2010), and the most recent phylogenomic studies on pancrustaceans add new transcriptome data on several crustaceans ( von Reumont et al., 2012 and Oakley et al., 2013). The number of sampled crustacean groups remains low, with the majority of EST data
representing economically important species, such as Antarctic krill Euphausia superba ( De Pittà et al., 2008), Pacific white shrimp Litopenaeus vannamei ( Gorbach et al., 2009 and Li et al., 2012), porcelain crab Petrolisthes cinctipes ( Stillman et al., 2006 and Tagmount find more et al., 2010) and, Daphnia pulex ( Colbourne et al., 2012). The main goal of the present study was to generate a preliminary EST databank of P. pollicipes. We constructed an in-house developed EST library which was merged with the EST data obtained by Meusemann et al. (2010). In our view, this databank will be highly useful in further studies focusing on specific genes, e.g. secreted proteins of the cement glands, or more generally to provide useful genomic information on P. pollicipes. Adult P. pollicipes were collected from O Roncudo Selleck Ixazomib (43° 15′ 51″N, 8°
58′ 45″W) (Corme, Galicia, Spain) and stored in RNAlater® (Life Technologies). Total RNA was extracted with Aurum™ Total RNA Mini Kit (BioRad) from part of the foot tissue. The CreatorTM SMARTTM cDNA Library Construction Kit (Clontech) was used with minor adaptations to create a cDNA library with full-length insertion. The cDNA was ligated to pDNR-LIB vector and then transformed into Escherichia coli (TOP10). Recombinant white colonies were randomly picked out and amplified by PCR using M13 primers.
PCR products were visualized on 1% agarose gels to ensure quality of amplification. Amplicons were directly sequenced however after being purified with ExoSAP-IT (USB). Sequencing reactions were carried out using both M13 Forward and M13 Reverse primers in a capillary DNA sequencer (3130xl Genetic Analysis System, Applied Biosystems). Electropherograms were quality controlled using Geneious Pro 5.4.6 (Drummond et al., 2011). Finally 119 high-quality ESTs remained for analysis and were deposited in GenBank (accession nos. HG792878–HG792996). In addition to this, 4191 ESTs of P. pollicipes ( Meusemann et al., 2010) were included into analysis (accession nos. FN243242–FN247432). Both EST sets were assembled into unigenes using the iAssembler software ( Zheng et al., 2011) that employs MIRA ( Chevreux et al., 2004) and CAP3 ( Huang and Madan, 1999) to generate initial assemblies. The individual ESTs were assembled into unigenes including contigs and singlets with minimum overlap of 30 bp and minimum percent identity of 97. Resulting unigenes were translated into amino acids following the pipeline described in von Reumont et al. (2013). BLAST2GO software ( Conesa et al., 2005) was used to conduct BLAST (nr database, blastP, e-value = 0.