GSM6246306: EBF4-FLAG Jurkat T cell ChIP rep1; Homo sapiens; ChIP-Seq
FLAG tagged EBF4 Jurkat T cells were isolated by EGFR biotinylated antibody (R&D) and were cross-linked in 1% formaldehyde for 15 minutes at room temperature, quenched in 2.5 M glycine for 5 minutes, washed, and resuspended in 1st lysis buffer (50 mM HEPES-KOH, 140 mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% TritonX-100, and proteinase inhibitor cocktail) and incubated 10 minutes. The cells were resuspended with 2nd lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, and proteinase inhibitor cocktail) and incubated 10 minutes. Cells were resuspended with 3rd lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritonX-100, 0.1% Na-Deoxycholate, 0.1% SDS, and proteinase inhibitor cocktail) and incubated 10 minutes. Cross-linked chromatin was sonicated for 4 minutes using a Biruptor-Pico (30 seconds on/30 seconds off). Precleared chromatin extract was incubated overnight at 4°C with anti-FLAG antibody (Cell signaling) and immunoprecipitated with protein G sepharose beads. DNA was used to generate a standard Illumina sequencing library.