Fluorescence-Activated Cell Sorting and Gene Expression Profiling of GFP-Positive Cells from Transgenic Zebrafish Lines
Gene expression profiling is a useful approach for deeper understanding of the specificity of cells, tissues, and organs in the transcriptional level. Recent development of high-throughput next-generation sequence (NGS) allows the RNA-seq method for this profiling. This method provides precise information of transcripts about the quantitation and the structure such as the splicing variants. In this chapter, we describe a method for gene expression profiling of GFP-positive cells from transgenic zebrafish by RNA-seq. We labeled specific cells in the brain with GFP by crossing a Gal4 driver line with the UAS:GFP line, isolated those cells by fluorescence-activated cell sorting (FACS), and analyzed by RNA-seq.
Key wordsTol2 transgenesis Gene trap Enhancer trap Gal4-UAS system GFP FACS RNA extract RNA-seq Next-generation sequence Gene expression analysis
We thank Naoyuki Inagaki for the helpful advice about cell dissociation methods. This work was partly supported by the National BioResource Project (to KK), Grant-in-Aids for Scientific Research on Innovative Areas “Genome Science” (221S0002 to YS), and Grant-in-Aids for Scientific Research (A) (23241063 and 15H02370 to KK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
- 2.Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K (2008) Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like. Development 135:159–169CrossRefPubMedGoogle Scholar
- 16.Wang K, Singh D, Zeng Z, Coleman SJ, Huang Y, Savich GL, He X, Mieczkowski P, Grimm SA, Perou CM et al (2010) MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res 8:1–14Google Scholar
- 17.Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515CrossRefPubMedPubMedCentralGoogle Scholar
- 18.Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ, Gnirke A, Nusbaum C et al (2010) Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat Biotechnol 28:503–510Google Scholar