Computational protocol: Transcriptomic Analysis of Purified Embryonic Neural Stem Cells from Zebrafish Embryos Reveals Signaling Pathways Involved in Glycine Dependent Neurogenesis

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Protocol publication

[…] Two independent clutches were injected, by each of two experimenters, corresponding to experimental duplicates. Total RNA from RNAlater-fixed embryos (Ambion) was extracted using RNAsolv reagent (Omega Biotek) following the manufacturer’s standard protocol. RNA extraction was made using between 5.19–9.75 × 105 cells by RNAsolv reagent manufacturer’s protocol (Omega Biotek). Absence of contamination with chemicals was assessed by nanodrop using 260/280 and 260/230 ratios. Quantification of total RNA was made by nanodrop and 0.1 to 1.44 g of total RNA was used for sequencing. Quality of total RNA was assessed with the BioAnalyzer Nano (Agilent) and all samples had a RNA Integrity Number (RIN) above 8.Library preparation was done with the Truseq RNA (Illumina). 18 PCR cycles was required to amplify cDNA libraries. Libraries were quantified by nanodrop and BioAnalyzer. All librairies were diluted to 10 nM and normalized with the Miseq SR50 v2. Libraries were pooled to equimolar concentration and multiplexed by six samples per lane. Sequencing was performed with the Illumina Hiseq2000 using the SBS Reagent Kit v3 (100 cycles, paired-end) with 1.6 nM of the pooled library. Cluster density was targeted at around 800 k clusters/mm2. Around 65 to 89 million reads were generated for each sample. Library preparation and sequencing was done at the Institute for Research in Immunology and Cancer’s Platform (University of Montreal). About 86% of high quality reads were mapped onto the zv9 version of the zebrafish genome (ensemble release 77) using TopHat version 2.0.10.Differential gene expression analysis was assessed by DeSeq2 package using R software. Differential gene expression was filtered on an absolute value of LogFC > 1 and a False Discovery Rate (or adjusted p-value) < 0.05. Pathway analysis was performed using DAVID bioinformatics resources (). The list of differentially expressed genes was uploaded onto DAVID analysis wizard and a list of all expressed genes found in our dataset was used as a background for gene enrichment analysis. We also benefitted form a free trial of the Ingenuity Pathway Analysis software from QIAGEN. Pathway information was generated using the Kyoto Encyclopedia of Genes and Genomes (KEGG: www.genome.jp/kegg/pathway). […]

Pipeline specifications

Software tools TopHat, DESeq2, DAVID, IPA
Databases KEGG KEGG PATHWAY
Organisms Danio rerio
Chemicals Calcium, gamma-Aminobutyric Acid, Glycine