Computational protocol: Molecular and Cellular Characterization of a Zebrafish Optic Pathway Tumor Line Implicates Glia-Derived Progenitors in Tumorigenesis

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Protocol publication

[…] RNA-Seq data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2886. Tissues for isolation of total RNA for RNA-Seq libraries were dissected from three age-matched genotypes. Total RNA was isolated using Qiagen RNeasy RNA Isolation Kit (Qiagen). The first sample, “Wild Type”, contained three pooled retinas from 6-month-old wild type adults. The second sample, “Pretumor”, consisted of three pooled retinas from age-matched heterozygous Tg(flk1:RFP)is18/+ adults that did not show obvious gross ocular tumors. The third sample, “Tumor”, consisted of tumor tissue dissected from the eyes of two age-matched heterozygous Tg(flk1:RFP)is18/+ adults that had advanced, large tumors that filled the vitreous and distorted the ocular cavity. RNA-Seq libraries from each sample were 100 bp paired-end sequenced in individual lanes on an Illumina HiSeq 2000 instrument at the Genome Sequencing and Analysis Core Resource, Duke Institute for Genome Sciences and Policy, Duke University. Mapping of the RNA-Seq data was performed at the Genome Informatics Facility, Iowa State University. To map the RNA-Seq data the Danio rerio reference genome v9 and gene models (release 71) were downloaded from ensemble. Sequences were mapped using GSNAP version 2012-07-20 . Raw read counts per gene were determined using the program HTSeq-count (http://www-huber.embl.de/users/anders/HTSeq/) to identify unique reads that mapped within a gene model. Upper quartile normalization was applied to the raw reads across the three samples. The Fisher's exact test was performed to determine differential expression of a gene between samples. Q-value estimation for false discovery rate was performed in R using open source software qvalue at bioconductor (http://bioconductor.org/biocLite.R; Alan Dabney, John D. Storey and with assistance from Gregory R. Warnes. qvalue: Q-value estimation for false discovery rate control. R package version 1.32.0). Heat maps representing log2(fold change) in gene expression were created in Excel. GO Term analysis was done at the Gene Ontology (GO) Tools website http://go.princeton.edu.Two-Step real time PCR was performed on a Roche LightCycler 480 instrument using SYBR green reaction mix (Fisher). RNA isolation from Wild Type, Pre-tumor, and Tumor tissues was as described above. cDNA template was synthesized with SuperScript II (Invitrogen). qRT-PCR reactions were run in triplicate for each tissue template and primer pair. Primers for qRT-PCR are listed in . […]

Pipeline specifications

Software tools GSNAP, HTSeq
Application RNA-seq analysis
Organisms Danio rerio
Diseases Glioma, Neoplasms, Retinal Neoplasms