Total RNA was extracted and purified from the thymus of WT and IL2RG KO pigs using RNeasy columns (Qiagen; Valencia, CA, USA) according to the manufacturer’s protocol. The RNA quality was verified using an Agilent Bioanalyzer 2100 (Agilent Technologies; Palo Alto, CA, USA). Generation of double-stranded cDNA, preparation and labeling of cRNA, hybridization to Agilent SurePrint HD Porcine GE Microarrays 4x44K (v2) Microarry (Agilent®) , washing, and scanning with Agilent Microarray Scanner D (Agilent Technologies, Inc.).
Label
Cy3
Label protocol
RNA labeling and hybridization were performed by using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, V 6.5, 2010). Briefly, 200ng of total RNA from each sample was linearly amplified and labeled with Cy3-dCTP. The labeled cRNAs were purified by RNAeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000(NanoDrop, Wilmington, USA).
Hybridization protocol
1650ng of each labeled cRNA was fragmented by adding 11 μl 10 x blocking agent and 2.2 μl of 25 x fragmentation buffer, and then heated at 60°C for 30 min. Finally 55 μl 2 x GE hybridization buffer was added to dilute the labeled cRNA. 100μl of hybridization solution was dispensed into the gasket slide and assembled to the Agilent SurePrint HD Porcine GE 4X44K Microarrays (Agilent®). The slides were incubated for 17 h at 65°C in an Agilent hybridization oven. then washed at room temperature by using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, V 6.5, 2010).
Scan protocol
The hybridized array was immediately scanned with an Agilent Microarray Scanner D (Agilent Technologies, Inc.)
Description
Replicate 1
Data processing
Raw data were extracted using the software provided by Agilent Feature Extraction Software(v11.0.1.1). The raw data for same gene was then summarized automatically in Agilent feature extraction protocol to generate raw data text file, providing expression data for each gene probed on the array. Array probes that have Flag A in samples were filtered out. Selected gProcessedSignal value was transformed by logarithm and normalized by quantile method.
