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Pipeline publication

[…] daptor P7, while sense primer Oligo(dG)-adaptor P8 and antisense primer 5R1~8 were used to get the 5’ end according to the Usage Information of 5’ RACE system (Invitrogen). The obtained PCR products were purified and sequenced as described above. The sequences were overlapped, verified, and subjected to cluster analysis., The homology searches of the cDNA sequence and amino acid sequence of CfNOS were conducted with BLAST algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.gov/blast). The deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org). The presence and location of signal peptide was predicted using the SignalP 3.0 program (http://www.cbs.dtu.dk/services/SignalP). The protein domains were predicted with the simple modular architecture research tool (SMART) version 7.0 (http://www.smart.emblheidelberg.de/). The multiple sequence alignment of CfNOS with other NOSs was performed with the ClustalW multiple alignment program (http://www.ebi.ac.uk/clustalw/) and displayed through the multiple alignment show program (http://www.bio-soft.net/sms/index.html). An unrooted phylogenic tree was constructed based on the deduced amino acid sequence of CfNOS and other NOSs by the Maximum Likelihood (ML) algorithm using the Seaview 4.0 software [,]., The quantitative real-time PCR amplification was carried out in a total volume of 25.0 µL, containing 2.0 µL 100 × diluted cDNA, 12.5 µL 2 × SYBR Premix Ex TaqTM II (Applied Biosystems, USA), 0.5 µL of each primers (10 mmol L-1) and 9.5 µL DEPC-water. For CfNOS, a 91 bp product was amplified with the primers P1 and P2 (. Two β-actin primers and two CfEF-1α (elongation factor 1 alpha from scallop C. farreri) primers ( were used to amplify 94 and 86 bp fragments, respectively, as internal controls to verify the successful reverse transcription and calibrate the cDNA template. The β-actin was referenced in the tissue distribution analysis and stimulation experiments, […]

Pipeline specifications

Software tools SignalP, Clustal W, SeaView
Databases ExPASy