strain background: AB. Genotype: cd41-GFP transgenic, isolated GFP positive cells Age: 35 hours post fertilization
Gerhard J. Weber
gata1-GFP+/+(12 somites), lmo2-GFP+/+ (12 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf)2 cells were separated from GFP-/- cells by flow cytometry at the indicated stages. Sorting was based on propidium iodide exclusion, forward scatter, and GFP fluorescence, using a FACSVantage flow cytometer (Beckton Dickinson). Sorted cell populations were run twice to optimize cell purity. Total RNA from cell-sorted populations was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy resins (Qiagen) according to the manufacturer’s recommendations. Total RNA was subjected to two rounds of linear amplification (Ambion) and hybridized to Affymetrix zebrafish Gene Chips, according to Affymetrix guidelines. After staining with a streptavidin-phycoerythrin conjugate (Molecular Probes), the fluorescence of bound RNA was quantitated by using a Gene Chip scanner (Affymetrix). The raw expression data were calculated and, after pairing of GFP+/+ and GFP-/- samples, statistically analyzed using methods implemented in Bioconductor’s “affy” package and available custom scripts 3,4. references: 1. Zhu, H. et al. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Dev Biol 281, 256-69 (2005). 2. Lin, H. F. et al. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 106, 3803-10 (2005). 3. Choe, S. E., Boutros, M., Michelson, A. M., Church, G. M. & Halfon, M. S. Preferred analysis methods for Affymetrix GeneChips revealed by a wholly defined control dataset. Genome Biol 6, R16 (2005). 4. Weber, G. J. et al. Mutant-specific gene programs in the zebrafish. Blood 106, 521-30 (2005).
Embryos were obtained by pairwise matings of adult transgenic fish, raised at 28 ºC.
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Following fragmentation, 15 microg of cRNA were hybridized for 16 hr at 45C on GeneChip Zebrafish Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
GeneChips were scanned using the Affymetrix GeneChip Scanner.
Gene expression data from sorted zebrafish GFP positive cell populations at 35hours post fertilization.
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.