Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. RNA quality was checked by agarose gel electrophoresis and RNA quantity was determined by spectrophotometry at 260 nm. RNA was then further purified using the RNeasy kit (Qiagen, Valencia, CA). The total RNA was used for gene expression analysis on an Affymetrix GeneChip Chicken Genome Array (Affymetrix, Santa Clara, CA), containing 38,535 probes.
Label
biotin
Label protocol
cDNA was synthesized using the One-Cycle cDNA Synthesis Kit (Affymetrix). Single-stranded (ss) cDNA was synthesized using Superscript II reverse transcriptase and T7-oligo (dT) primers at 42°C for 1 h. Double-stranded (ds) cDNA was obtained using DNA ligase, DNA polymerase I, and RNase H at 16°C for 2 h, followed by T4 DNA polymerase at 16°C for 5 min. After cleanup using a Sample Cleanup Module (Affymetrix, Santa Clara, CA), ds cDNA was used for in vitro transcription (IVT). cDNA was transcribed using the GeneChip IVT Labeling Kit (Affymetrix) in the presence of biotin-labeled CTP and UTP.
Hybridization protocol
the biotin-labeled IVT-RNA was fragmented and hybridized to the chicken genome GeneChip array at 45°C for 16 h, according to the manufacturer’s instructions.
Scan protocol
After hybridization, the arrays were washed in a GeneChip Fluidics Station 450 with a non-stringent wash buffer at 25°C, followed by a stringent wash buffer at 50°C. After washing, the arrays were stained with a streptavidin–phycoerythrin complex. After staining, intensities were determined with a GeneChip scanner, controlled by GeneChip Operating Software (GCOS; Affymetrix).
Description
GSCs separated by MACS treatment from the gonad of 6.5 days WL embryos.
Data processing
The quality of the array image was assessed as described in the GeneChip expression analysis manual (Affymetrix). All arrays were processed to determine the “robust multi-array average” (RMA) using the “affy” software package (Irizarry et al., 2003; Gautier et al., 2004). Expression values were computed in detail from raw CEL files by applying the RMA model of probe-specific correction for perfect-match probes. These corrected probe values were then subjected to quantile normalization, and a median polish was applied to compute one expression measure from all probe values. Resulting RMA expression values were log2 transformed.
