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Protocol publication

[…] 1.2% agarose gel, and quantified using a Nanodrop spectrophotometer (Thermo scientific). Total RNA samples were sent to Australian Genome Research Facility (Brisbane, Australia) for library construction and sequenced (paired-end) using an Illumina HiSeq 2500 sequencing platform. Raw sequence reads (100 bp) were assembled into contigs (>200 bp) using the CLC genomics software (Qiagen). Protein coding regions were determined using the open reading frame (ORF) predictor. Relative expression of genes in each tissue transcriptome was determined based on RPKM (Reads Per Kilobase of exon per Million mapped reads) values, utilizing the commercially available CLC Genomic Workbench 7 software., A BLASTp search was used to annotate proteins from each C. tritonis transcriptome. Schematic diagrams of protein domain structures were prepared using IBS illustrator (IBS, version 1.0) software. Multiple sequence alignments were performed using the MEGA 6.0 platform with the ClustalW protocol and the Gonnet protein weight matrix. SWISS-MODEL was used to predict the 3D protein structure of an echotoxin-like protein identified from giant triton SG. First, BLASTp analysis was used to identify a template that shared significant sequence similarity to a C. tritonis echotoxin sequence. The best match was selected based on the presence of similar domains and plausibility quality control. Finally, based on the alignment, the coordinates of the model were constructed for the structurally conserved regions of the model. N-terminal signal sequences were predicted using the SignalP 4.1, Predisi and TMHMM. A protein was designated as secreted only when it met the criteria of […]

Pipeline specifications

Software tools BLASTP, MEGA, Clustal W