Fragmentation buffer

Labeling of isolated RNA was done using the Low Input Quick Amp Labeling kit (Agilent Technologies, Palo Alto, CA, USA), according to the manufacturer’s instructions. For whole-genome expression analysis, samples were hybridized to Agilent 4x44K v2 Whole Human Genome arrays (G4845A; Agilent Technologies), covering 27,958 genes. In brief, equal amounts of total RNA (50 ng) were amplified and labeled with either Cy3-CTP (experimental samples) or Cy5-CTP (reference material, obtained as described above) using the Low Input Quick Amp Labeling kit (Agilent Technologies). For hybridization, equal amounts (825 ng) of labeled samples were fragmented in Fragmentation Buffer (Agilent Technologies) for 30 min at 60 °C. Labeled and fragmented complementary RNA (cRNA) was hybridized to the array and incubated in a rotating hybridization chamber for 17 h at 60 °C. After hybridization, the array was washed subsequently for 5 min in 6 x saline sodium phosphate-EDTA (SSPE)/0.005% N-lauroylsarcosine, 1 min in 0.006 x SSPE/0.005% N-lauroylsarcosine, and 30 s in acetonitrile and dried quickly in nitrogen flow. The arrays were scanned at a resolution of 5 mm and at 5 and 100% photomultiplier tube settings using the Agilent DNA Microarray Scanner (Agilent Technologies). Scan data were extracted using Agilent Feature Extraction software (version 8.5.1; Agilent Technologies).

RNA was extracted from the spleen and thymus of mIL2RG^+/Δ69-368 KO and WT pigs using RNeasy Mini Kits (Qiagen, Valencia, CA, USA) according to the manufacturer protocol. We used the Porcine (V2) Gene Expression Microarray, 4×44K (G2519F). Labelling was carried out using an RNA Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, USA; http://www.agilent.com). The sample and control RNAs were labelled with Cy-3 and Cy-5, respectively. Fragmentation was carried out by incubation at 60°C for 30 min in fragmentation buffer (Agilent Technologies), and the process was stopped by the addition of an equal volume of hybridisation buffer (Agilent Technologies). The fragmented target was applied to an Oligo Microarray for pig (Agilent Technologies). Hybridisation was carried out at 60°C for 17 h in a hybridisation oven (Robbins Scientific, Sunnyvale, CA, USA). The hybridised array was scanned with an Agilent microarray scanner. The TIFF image generated was loaded into Feature Extraction Software (Agilent Technologies) for feature data extraction, and data analyses were performed with GeneSpring 10.0. Microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE77581.

Microarray experiment procedures were carried out following the manufacturer’s protocols. Total RNA was amplified by an Agilent Quick Amp Labeling Kit (Agilent Technologies, USA). Cy-labeled cRNA (0.3 μg) was cleaved to an average size of about 50–100 nucleotides by incubation with fragmentation buffer (Agilent Technologies) at 60 °C for 30 min. Equal Cy-labeled cRNA was pooled and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarray (Agilent Technologies, USA), then scanned by an Agilent microarray scanner (Agilent) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images were analyzed by Feature Extraction software 10.5 (Agilent Technologies), and image analysis and normalization software was used to quantify signal and background intensity for each feature, which substantially normalized the data using the rank-consistency-filtering LOWESS method.