GSM5531003: KO-NAD-WWE (ChAP-seq); Homo sapiens; OTHER
isolated mitochondria were cross-linked in a solution of 4 % formaldehyde in PBS for 10 min on ice The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. Mitochondrial pellets were resuspended in buffer B (100 mM Tris–Cl (pH 8.1), 1 % sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitor cocktail) and the chromatin was sheared with S-450 sonicator (Branson, Danbury, CT USA) or Bioruptor Pico (Diagenode, Denville, NJ USA). Sonication conditions were optimized by analyzing purified DNA samples on the BIOANALYZER (Agilent, DNA1000 Kit). The prepared mtDNA fraction was diluted 1/10 in IP buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–Cl (pH 8.1), 167 mM NaCl and a protease inhibitor cocktail) and incubated with PARP1 (39559, Active Motif, Carlsbad, CA USA) or TFAM (ABE483, Millipore-Sigma) antibodies, overnight at 4 °C. Samples were incubated for 1 h at 4 °C with protein A or G beads. Then the beads were washed with TSE150 (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–Cl (pH 8.1), 150 mM NaCl), TSE500 (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–Cl (pH 8.1), 500 mM NaCl), Buffer III (0.25 M LiCl, 1 % NP-40, 1 % sodium deoxycholate, 1 mM EDTA, 10 mM Tris-Cl (pH 8.1)) and two times with TE (pH 8.0) for 10 min in each solution. Bead-bound chromatin was eluted with elution buffer (1 % SDS, 0.1 M NaHCO3 (pH 8.0)) for 30 min at 65 °C. The supernatant containing the chromatin, without the beads, was isolated and incubated overnight at 65 °C with 200 mM NaCl to reverse the cross-links. Five hundred microliters of the sample were incubated at 50 °C after adding 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris (pH 6.5) and 4 μl of Proteinase K (20 mg ml-1) and then purified with phenol/chloroform/isoamyl alcohol. Nucleic acids were precipitated by centrifugation for 30 min at 4 °C after mixing the sample with 1 μl of glycogen solution (20 mg ml-1), 20 μl 5 M NaCl and 500 μl isopropanol. Purified nucleic acid pellets were washed with 70 % ethanol, dried and dissolved in nuclease-free water. According to the manufacturer's protocol