Chip dna purification kit

The ChIP assay was performed with a commercial kit (Beyotime Institute of Biotechnology; cat. no. P2078) according to the manufacturer's instructions. Briefly, cells were cross-linked using 1% formaldehyde (Sigma-Aldrich; Merck KGaA) in PBS at 25°C for 10 min. Termination of cross-linking was achieved by the addition of glycine (Beijing Solarbio Technology Co., Ltd.) to a final concentration of 125 µM. Then, 1×10⁶ cells were collected via centrifugation at 300 × g for 3 min at 25°C and washed twice with pre-chilled PBS. The cells (1×10⁶) were treated with 2 µg anti-HOXD9 (Santa Cruz Biotechnology, Inc.; cat. no. sc-137134), anti-SCNN1A (Abcam; cat. no. ab272878) or anti-mouse IgG (Beyotime Institute of Biotechnology; cat. no. A7028) antibodies, which were immunoprecipitated against cross-linked 100 µg DNA (using a spectrophotometer at 260 nm)/protein and incubated at 4°C for 2 h. A ChIP DNA purification kit (Beyotime Institute of Biotechnology; cat. no. D0033) was used to purify the immunoprecipitated DNA and it was amplified via qPCR as described above. RT-qPCR was applied to the analysis of the enriched DNA.

A ChIP assay kit (Beyotime Institute of Biotechnology) was used to perform the ChIP-seq analysis, according to the manufacturer's instructions. Briefly, HUVECs were fixed with 1% formaldehyde for 10 min at room temperature. Next, 1x10⁶ HUVECs were collected via centrifugation at 300 x g for 3 min at 25˚C and washed twice with phosphate-buffered saline. Subsequently, HUVECs were lysed RIPA buffer (Cell Signaling Technology, Inc.) and sonicated for 30 min. The sonicated cell lysates (2 µl) were immuno-precipitated with 4 µg antibodies against FOXP1 (1:800; cat. no. ab134055; Abcam) or IgG (negative control, 1:800; cat. no. ab109489; Abcam) at 4 ˚C overnight. The next day, the samples were conjugated with Protein A agarose (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h at 4˚C. Finally, the immunoprecipitated DNAs were purified with a ChIP DNA purification kit (Beyotime Institute of Biotechnology) and amplified by virtue of qPCR as aforementioned. RT-qPCR was applied to analyze the enriched DNA. The primers used for the ChIP assay were the same as those used in RT-qPCR.

The EZ CHIP kit (Shanghai Huzheng Biotechnology Co., Ltd.) was employed for the CHIP assays. The cross-linking of the cells was performed with 1% formaldehyde, whereas their quenching with 2.5 M glycine. Then, 1x10⁶ cells were collected via centrifugation at 300 x g for 3 min at 25˚C and washed twice with pre-chilled PBS. DNA was immunoprecipitated from the sonicated cell lysates using 2 µg anti-KAT5 (1:30; cat. no. ab300521; Abcam) at 4˚C overnight. The following day, the samples were conjugated with Protein A agarose (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h at 4˚C. A ChIP DNA purification kit (Beyotime Institute of Biotechnology) was used to purify the immunoprecipitated DNA and it was amplified by qPCR. RT-qPCR was applied to analyze the enriched DNA.