Dna purify kit

Nuclear proteins were prepared according to the ChIP protocol. 500 U of the BamHI restriction enzyme (TaKaRa, Japan) was added to the DNA-protein cross-linked sample and digested overnight at 37 °C. The next day, 10% SDS was added to the digested samples, which were incubated at 65 °C for 20 min to inactivate the restriction enzyme. The sample was diluted 1:10 using the DNA Ligation Kit (TaKaRa), incubated at 16 °C for 4.5 h, and further incubated at room temperature for 30 min. Next, 300 mg of proteinase K (Beyotime) and 300 mg of RNase A (Beyotime) was added to the sample and incubated at 65 °C overnight. Then digested DNA was purified and pelleted using the DNA Purify Kit (Millipore) and amplified by qRT-PCR.

ChIP was performed with a ChIP Assay Kit (Millipore) according to the manufacturer’s procedure. Briefly, 5 × 10⁶ cells were transfected in a 10 cm culture dish for 48 h, cross-linked using 1% paraformaldehyde for 10 min at room temperature, and incubated with glycine for 5 min to stop the cross-linking reaction. The fixed cells are harvested, lysed, and sonicated. The lysates were incubated with 50 µL of protein A/G magnetic beads (Bimake) and 5 µg of primary antibody on a rotator at 4 °C overnight. The DNA-protein cross-links were eluted and proteins were digested with proteinase K (Beyotime). The digested DNA was purified and pelleted using the DNA Purify Kit (Millipore) and amplified by qRT-PCR.