Computational protocol: Dietary Intake Influences Adult Fertility and Offspring Fitness in Zebrafish

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Protocol publication

[…] Gamete samples were collected from fish during the weight gain measurements that followed the dietary intervention. Anaesthetized females were blotted dry, placed in a dish, and pressed gently on the belly to release the eggs. The eggs were gathered with a spatula, transferred to tube containing RA1 buffer (Macherey-Nagel, cat. 740955.250) with β-mercaptoethanol, and stored at -80°C until RNA extraction.Total RNA was prepared from gamete samples taken from group 3 (dictated by the number of samples obtained from the 5 mg arm). The RNA was filtered with the NucleoSpin RNA kit (Macherey-Nagel, cat. 740955.250) and bound and eluted with columns (17–23,000 nt size range) from the RNA clean and concentrator kit (Zymo cat. no. R1017). The resulting RNA was checked for quality and quantity by Nanodrop, Qubit, and Bioanalyzer.Library preparation and RNA sequencing was performed by New Zealand Genomics Limited. Total RNA libraries were prepared for the egg samples using the TruSeq stranded total RNA library kit with Ribozero (Illumina). The libraries were run on the HiSeq 2500 (Illumina) to generate single-ended 100 bp reads. The sequence reads were analyzed using the Tuxedo suite []. BiNGO, a Cytoscape plugin, was used for gene ontology analysis [,]. For comparing diet-induced changes in gene expression with other datasets, the HCOP: Orthology Predictions Search was used to obtain the human orthologs []. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [] and are available through GEO series accession number GSE81007 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81007). […]

Pipeline specifications

Software tools Tuxedo, BiNGO
Databases GEO HCOP
Application RNA-seq analysis
Organisms Danio rerio
Diseases Malnutrition