GSM8045631: Ctrl_IP_DNA2; Homo sapiens; ChIP-Seq
Cells were harvested by scraping, washed twice with prechilled PBS, and fixed with 1% formaldehyde at room temperature for 10 minutes, and the reaction was then quenched with 0.125 M glycine. After being washed once with PBS, the cells were lysed with buffer 1 (50 mM HEPES, pH 7.5; 140 mM NaCl; 1 mM EDTA, pH 8.0; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100) and buffer 2 (200 mM NaCl; 1 mM EDTA; 0.5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA); 10 mM Tris-HCl, pH 8.0) at 4°C for 10 minutes. After centrifugation, the pellets were treated with lysis buffer (1% SDS, 10 mM EDTA, pH 8.0; 50 mM Tris-HCl, pH 8.0). All the above buffers contained 1 mM PMSF (Amresco), 0.6-2 μg/mL aprotinin (TargetMol) and 1× cocktail (CWBIO). The mixtures were subjected to sonication with a Covaris M220 focused ultrasonicator for 4 minutes to obtain smeared DNA fragments of 200-500 bp. After sonication, the samples were centrifuged at 15000 rpm, and the supernatants were diluted with an equal volume of NETN buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA (pH 8.0), and 0.5% NP-40). A portion of each sample was taken as input; 5 μg of an anti-DNA2 (Abcam, ab96488), or anti-MRE11 (Bethyl, A301-998A) was added to the remainder of each sample; and the samples were then incubated with rotation at 4°C overnight. Protein A (Millipore) beads were added to each sample, and the samples were incubated with rotation at 4°C for 2-3 hours. The beads were subsequently washed successively with the following buffers: one wash with low-salt buffer (0.1% SDS, 1% Triton X-100; 2 mM EDTA, pH 8.0; 20 mM Tris-HCl, pH 8.0; 150 mM NaCl); one wash with high-salt buffer (0.1% SDS, 1% Triton X-100; 2 mM EDTA, pH 8.0; 20 mM Tris-HCl, pH 8.0; 500 mM NaCl); one wash with LiCl buffer (10 mM Tris-HCl, pH 8.0; 1% sodium deoxycholate; 1% NP-40; 250 mM LiCl; 1 mM EDTA, pH 8.0); and two washes with TE buffer (10 mM Tris-HCl, pH 8.0; and 1 mM EDTA, pH 8.0). Then, the beads were incubated in 200 μL of elution buffer (10 mM Tris-HCl, 1% SDS, 1 mM EDTA) and incubated in a Thermo Mixer-Heat with vibration at 1200 rpm at 65°C overnight. Finally, the samples were incubated with 10 μg/mL RNase A at 37°C for 1 h and then with 20 μg/mL proteinase K at 55°C for 1 hour. The samples were extracted with phenol–chloroform. Then, 10 μL of glycogen (20 mg/mL), 100 μL of ammonium acetate (7.5 M), 40 μL of NaCl (5 M), and a 5x volume of ethanol were added to each supernatant and mixed, and the mixtures were incubated at -20°C overnight. The next day, the samples were centrifuged at 15000 rpm for 30 minutes at 4°C. We washed each pellet once with 75% ethanol and then centrifuged the sample and removed the supernatant. Finally, we resuspended the pellet in nuclease-free water to prepare the sample for sequencing. The purified DNA was used NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645L) for library preparation.