GSM9176664: WT anti-Helios; Mus musculus; ChIP-Seq
Tregs were purified using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) from the spleens of WT or Eos-flox x Foxp3-Cre (Eos cKO) mice and fixed for 10 min at room temperature with 1% Formaldehyde (Millipore Sigma). Cross-linking reaction was stopped by adding 125 mM Glycine (Millipore Sigma). Cells were washed twice in ice-cold PBS and cell pellets were flash-frozen and stored at -80 °C. Cross-linked DNA was fragmentated by sonication using Digital Sonicator (Diagenode). The lysates was incubated overnight at 4 °C with 50 uL Protein A/G magnetic beads (Thermo Fisher Scientific) that had been pre-incubated with 6 ug appropriate antibodies. Samples were washed, eluted, reverse cross-linked at 65 °C for 24 h, and purified using ChIP DNA Clean & Concentrator (Zymo Research). For each ChIP analysis, 5 x106 – 10 x 106 cells were used. Antibodies used were anti-Eos 18H2 (this study) and anti-Helios D8W4X (Cell Signaling). library was prepared using NEBNext Ultra II End repair/dA-Tailing Module (New England Biolabs) and NEBNext Ultra II Ligation Module (NEB#E7595S/L) according to manufacturer's instructions.