Computational protocol: Byssus Structure and Protein Composition in the Highly Invasive Fouling Mussel Limnoperna fortunei

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[…] To identify adhesion-related genes, total RNA of foot tissue samples was extracted using mirVana miRNA Isolation Kit (Ambion Inc, Austin, Tex, USA) following the manufacturer's instructions. RNA integrity was evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA with integrity number (RIN) ≥ 7 was used for downstream analyses. A cDNA library was constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to manufacturer's instructions and sequenced on the Illumina Sequencing Platform HiSeqTM 2500 (Illumina, San Diego, CA, USA). The sequenced results were spliced by TRINITY software (Grabherr et al., ) with paired-end method. TGICL software was used to remove redundancy and get a set of final unigene as reference sequences. Raw data of L. fortunei foot transcriptome has been deposited into Sequence Read Archive (SRA) in NCBI (accession number SRP125019). These obtained sequences were subsequently submitted to Basic Local Alignment Search Tool (BLAST) against NR database (ftp://ftp.ncbi.nih.gov/blast/db) and Swiss-Prot database (http://www.uniprot.org/downloads) for functional annotation. A cutoff E-value ≤ 1.0e−5 was used to identify the most representative annotation. Clusters of Orthologous Groups for Eukaryotic Complete Genomes (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation were also performed using previously described methods (Hou et al., ; Li S. G. et al., ) with the cutoff E-value ≤ 1.0e−5. Typical unigenes with high homologies to the reported adhesion-related proteins in this transcriptome were selected and compared with other bivalve species using homologous alignment methods (Guerette et al., ; DeMartini et al., ). Homologous alignments for foot proteins were conducted using BioEdit version 7.2.5 (Hall, ) and Jalview online bioinformatics tool (http://www.jalview.org/). Repetitive motifs were predicted by RADAR tool (https://www.ebi.ac.uk/Tools/pfa/radar/) and conserved domains were predicted by PROSITE tool (http://prosite.expasy.org/). [...] Expressions of foot protein genes were analyzed using the RT-qPCR method. Foot tissue samples were grinded in liquid nitrogen. Total RNA isolation, purification and quantification were conducted using methods by Li S. G. et al. (). PrimeScript™ RT Reagent Kit (Takara, Tokyo, Japan) was used to synthesize cDNA following the manufacturer's instructions. A 20-μL reaction containing 0.4 μM of each primer, 1.0 μL of cDNA, and AceQ qPCR SYBR® Green Master Mix (Vazyme, Nanjing, China) was carried out on a LightCycler® 96 System (Roche Diagnostics, Mannheim, Germany). Thermal program included 1 cycle of 95°C for 600 s; 40 cycles of 95°C for 10 s and 60°C for 30 s; 1 cycle of 95°C for 10 s, 65°C for 30 s and 95°C for 10 s. The LfACTIN gene was used as the internal reference (Uliano-Silva et al., ). For tissue-specific analyses, the adductor muscle was used as the control tissue. For gene expression profile analysis, foot tissues from pre-cultured mussels were used as the day 0 control. Relative expression levels of these genes were calculated by the 2−ΔΔCT method (Livak and Schmittgen, ). Specific primers for all the genes (Table ) were designed using Primer3 version 4.1.0 (http://primer3.ut.ee/). [...] Statistical analyses of data were obtained from three biological replicates and expressed as mean ± standard deviation (SD). Differences in gene expression levels and byssus numbers were accessed using one-way ANOVA with the significance level at *p < 0.05. Micrographs were processed in Adobe Photoshop CS4 11.0 software and figures were drawn using SigmaPlot version 12.5 (Systat Software, San Jose, CA, USA). […]

Pipeline specifications

Software tools Primer3, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Limnoperna fortunei
Chemicals Amino Acids, Dihydroxyphenylalanine