Flag ChIP-seq in Atf7ip KO mESCs expressin Flag-dFNIII ATF7IP
For cross-linked ChIP (X-ChIP) analysis, 1 x 107 cells were suspended in 1 mL PBS containing 10% FBS, and 62.5 L of 16% formaldehyde (methanol-free) was added to the cell suspension. After 10 min incubation at 25C, the cross-linking was quenched by the addition of 100 L of 2.5 M glycine. After centrifugation at 6,500xg for 3 min at 4C, the pellets were brie y washed with ChIP-lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40, and 1x protease inhibitor cocktail) and were then incubated with 500 uL of the ChIP-lysis buffer for 5 min on ice. After centrifugation at 6,500xg for 3 min at 4C, the pellets were re-suspended in SDS-Lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, and 1x protease inhibitor cocktail). After 10 min incubation on ice, 400 L of Triton-buffer (15 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1x protease inhibitor cocktail) was added to the suspensions. The cell lysates were then sonicated using a Bioruptor UCD-250 (Diagenode). After centrifugation at 14,000xg for 10 min at 4C, the supernatants were diluted with Triton buffer to 1100. We used 100 L or 1000 L of the lysates for input samples or IP samples, respectively. For IP, the lysates were incubated with antibody-conjugated magnetic beads (Dynabeads M-280 Sheep anti-mouse IgG or Dynabeads Protein G, Invitrogen) at 4C for over-night. After sequential washes with Low-Salt-buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), High-Salt-buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl-buffer (10 mM Tris-HCl, pH 7.5, 25 mM LiCl, 1 mM EDTA, 1%Deoxycholate, 1% NP-40) and TE, the bound DNA was recovered and then analysed by qPCR.