ChIP Assay for Transcription Factor Binding Analysis

ChIP assay were carried out as described previously^{16 (link)}. In brief, CD4⁺CD25⁻CD62L⁺ T cells were purified by magnet cell sorting (Miltenyi Biotec) and cultured at 1 × 10⁶/well in 24-well plates with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence or absence of 2 ng/ml TGF-β1 and 10 ng/ml IL-4. 24 h later, cells were collected and fixed with 1% formaldehyde at room temperature for 10 min and lysed in the lysis buffer (Diagenode). The lysate were sonicated and precipitate with anti-E2A (V-18, Santacruz), anti-GATA3 (B-10, Santacruz), control rabbit IgG (Abcam) or control mouse IgG (CellSignaling). ChIP DNA was analyzed by qPCR (Bio-Rad) with the following primers: IL-9 promoter-forward: 5′-GGC CCA GCA CAG AAC TGA AGA GC and reverse: 5′-AGG GTT TTT CCC GGT TTG AAG AGC. ChIP-sequencing was performed as described previously^{35 (link)}.