Microarray Analysis of Intestinal Eosinophils

Microarray analysis on sorted intestinal eosinophils was performed as previously described.^{56 (link)} Small intestinal eosinophils were sorted as DAPI⁻CCR3⁺SiglecF⁺CD45⁺CD4⁻CD8a⁻CD19⁻B220⁻SSC^high cells from 10 animals using FACS Aria (BD). Total RNA from sorted eosinophils was extracted by standard TRIzol RNA isolation (Invitrogen) and subsequently column-purified with an RNeasy Mini Kit (Qiagen). mRNA integrity was validated by the Agilent 2100 bio-analyzer (Agilent Technologies). Eosinophil mRNA was amplified and labeled with the WT-Ovation Pico RNA Amplification System (NuGen) and subjected to the GeneChip Mouse Gene ST 1.0 Array chip (Affymetrix). Microarray expression analysis was performed at Cincinnati Children’s Hospital Medical Center’s Chip Core facility, and expression data were analyzed by the software of Genespring GX 11 (Agilent Technologies). The Affymetrix raw expression values were filtered with the significance threshold of 400 as previously reported^{56 (link),57 (link)} and validated by the levels of eosinophil-specific major basic protein gene (also known as proteoglycan 2 [Prg2]) and eosinophil non-expressed glucagon (Gcg) (Fig. S12).