GSM7061345: ECh_Abcg2N_input_2_052721; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation was performed with ULI-NChIP protocol. 10,000 FACS isolated  ECs were used per reaction. Cells were treated with the nuclei extraction buffer (10 mM Tris-HCl pH8.5, 140 mM NaCl, 5 mM  MgCl2, 0.6% NP40, 1x protease inhibitor cocktail) and chromatin was fragmented for 10 min using  MNase at 25°C and diluted in NChIP immunoprecipitation buffer (10 mM Tris-HCl pH8.0, 2 mM EDTA, 90 mM NaCl, 0.1% Triton X-100, 0.1% DOC, 1x protease inhibitor cocktail). Chromatin was incubated with 1μg of antibody bound to 11μl of protein A Dynabeads (Thermo Fisher Scientific, 10002D) and rotated overnight at 4°C. Then it was washed twice with 200 μl low-salt wash buffer (20 mM Tris-HCl pH8.0, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.1% SDS), and twice with 200μl high-salt wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA, 500mM NaCl, 1% Triton X-100, 0.1% SDS). DNA was eluted from chromatin in 100μl hot elution buffer (100mM NaHCO3, 1% SDS) for 2 hr at 65°C, then purified by phenol:chloroform and precipitated by  isopropanol overnight at -20°C. The DNA was resuspended in 10mM Tris-HCl pH 8.5. ChIP was  performed using the following antibodies: anti-H3K4me3 (Cell signaling Technology, 9727), anti-  H3K27me3 (Millipore, 07-449). ChIP sequence libraries were generated using the KAPA Hyper Prep Kit