GSM5399167: MED4_degron_6h_PolII_ChIP_Rep2; Mus musculus; ChIP-Seq
Cells were harvested at 70%–80% confluency by trypsinization. After washing with DPBS, cells were formaldehyde-crosslinked to a final concentration of 1% for 10 min at room temperature, followed by 5 min quenching with 125 mM glycine. Cells were washed twice with cold DPBS. If not used immediately, the cell pellet was flash frozen in liquid nitrogen and stored at −80°C. Crosslinked cells were resuspended in swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, and 1×cOmplete protease inhibitor cocktail) and incubated for 10 min on ice. Cells were centrifuged and the cell pellet was resuspended in Buffer A (50 mM HEPES-KOH pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) plus 1×cOmplete protease inhibitor cocktail. Cell sonication was performed on a Qsonica Q800R2 sonicator with 20% amplitude for 20 cycles at 10 s each with 30 s between cycles at 4°C. Sonicated lysate was pre-cleared by incubating with Dynabeads Protein A/G. Part of the pre-cleared lysate was used as input and the remainder was incubated overnight at 4°C with 2-10 μg of antibody. DNA/Protein-antibody conjugates were precipitated using Dynabeads Protein A/G. Beads were washed twice each with Buffer A, Buffer B (50 mM HEPES-KOH pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), LiCl buffer (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Na-deoxycholate, 0.5% NP-40) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). DNA was eluted in elution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS). Cross-links were reversed overnight at 65°C. RNA and protein were digested using RNase A and Proteinase K, respectively, and DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP-seq libraries were constructed using KAPA HyperPrep Kit.