ChIP was performed as described previously (18, 48). Briefly, MEF cells expressing TDP1 variants (1 × 107) were treated with mito-SN38 (5 μM) for 6 hours followed by fixation with formaldehyde (1.1% final concentration) for 10 min at room temperature, and reactions were stopped by adding 125 mM glycine for 5 min. Cells were then washed with phosphate-buffered saline (PBS), scraped, and lysed in 0.5 ml of ChIP lysis buffer [50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8), 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 1× protease inhibitor] on ice for 30 min. Lysates were sonicated 10 times for 30 s at 30% power to yield fragments of 200 to 300 bp and cleared by centrifugation at 14,000g for 10 min at 4°C. The supernatant (50 μl) was stored at −20°C as input and labeled as “no-IP.” The remainder was diluted fourfold in dilution buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA (pH 8), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1× protease inhibitors] and incubated with 10 μl of FLAG antibody on a rotating platform overnight at 4°C. The following day, 150 μl of A/G PLUS-Agarose beads (Santa Cruz Biotechnology) was added and further incubated on a rotating platform for 2 hours at 4°C. The beads were then washed twice with low-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 150 mM NaCl], twice with high-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 500 mM NaCl], followed by one wash with lithium chloride buffer [0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM tris-HCl (pH 8)], and twice with TE buffer. The immunoprecipitated complex was then eluted from the beads in 150 μl of elution buffer (1% SDS and 100 mM NaHCO3). The eluent and the no-IP control samples were then treated with RNase A and proteinase K. The DNA was purified using phenol-chloroform extraction and ethanol precipitation. The pellets were resuspended in 30 μl of distilled water.

For qPCR, the no-IP or input and ChIP samples were diluted 1:10; then, 5 μl was mixed with 2.5 μl of 5 μM forward and reverse primers (table S 1) with 10 μl of 2× SYBR Green PCR mix (Applied Biosystems). The PCR mix was aliquoted into reaction volumes (20 μl) in triplicate and amplified using an ABI 7500 thermocycler (Applied Biosystems). The PCR reactions were carried out with thermocycling conditions of 95°C for 10 min and then 40 cycles at 90°C for 15 s, 50°C for 15 s, and 72°C for 30 s, with signal acquisition at the end of each cycle. Quantification of chromatin enrichment was calculated by percent input normalized to the mtDNA copy number in the input samples. The sequences of the primers are listed in table S1.

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