title: Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing.
abstract: We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post-fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA.
authors.forename: Sataree
authors.initials: S
authors.lastname: Khuansuwan
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA. Electronic address: josh.gamse@vanderbilt.edu.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Gamse
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D017403:In Situ Hybridization; D010870:Pineal Gland / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*; D020825:T-Box Domain Proteins / Q000235:genetics; D013997:Time Factors; D059467:Transcriptome / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D020825:T-Box Domain Proteins; D029961:Zebrafish Proteins; C120107:tbx2b protein, zebrafish
keywords: Bioinformatics; Cell dissociation; Epithalamus; FACS; Parapineal; Pineal; Pineal complex; RNA-seq; Tbx2b; Transcriptome analysis; Zebrafish