ChIP assay was performed according to manufacturer’s protocol (Beyotime, Shanghai, China). DNA was extracted by DNA Purification Kit (Beyotime, Shanghai, China) and eluted for PCR. Primer sequences were listed in Supplementary materials .
Chip assay
Manufactured by Beyotime
Sourced in China
The ChIP assay is a laboratory technique used to investigate the interaction between proteins and DNA. It allows researchers to identify specific DNA sequences that are bound by a particular protein of interest within the cell.
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9 protocols using chip assay
1
ChIP Assay and DNA Purification
2
Mapping NF-κB binding to SOX4 promoter
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In all, 1 × 107 MDA-MB-231 or MCF-7 cells were collected and administrated with 1% formaldehyde for 15 min at room temperature. In total, 0.125 M glycine was added into the system for 5 min. Cells were subsequently scraped and centrifuged at 1000 g for 3 min at 4 °C. CHIP assay was conducted according to the manufacturer’s instruction (Beyotime, Nantong, China) by immune-precipitating the DNA targets with NF-κB antibody. The anti-human IgG was set as negative control. The −817 to −806 promoter region of SOX4 was predicted as the binding site of NF-κB by JASPAR database. The region was amplified from the DNA samples using the primer pair: forward 5′-TTACGGAGCACTACCTAATGTG-3′ and reverse 5′-CCTGTAAATCCTGCATAGCC-3′. The PCR products were then subjected to gel electrophoresis and compared between groups.
3
ChIP Assay for KLF4-miR-155-5p Binding
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The binding affinity of KLF4 and miR-155-5p was determined by ChIP assay based on the protocol (Beyotime). KLF4 level was increased or decreased by oe-KLF4 or sh-KLF4 vectors, respectively. HK-2 cells (1 × 106 cells) were processed by ultrasound for 48 cycles, after which cell supernatant was extracted by centrifugation. Followed by that, the beads were reacted with the target protein antibody or immunoglobulin G (IgG). The antibody-bound beads were incubated with the sample to bind the antibody to the target protein. KLF4 chromatin complex was immunoprecipitated by the anti-KLF4 antibody and then the target protein was eluted. In the experiment, anti-IgG (Santa Cruz, CA, USA) served as a control.
4
Regulation of MAOA Promoter by BAP31 and R1
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HEK-293T cells were co-transfected with shRNA-Bap31 and R1 over-expression plasmids or Bap31 overexpression plasmids and shRNA-R1 plasmids for 48 h. ChIP assay was performed according to the manufacturer’s protocol (Beyotime). The ChIP primer (Table 1 ) sets were checked for linear amplification and designed to amplify the region of the human MAOA promoter.
5
GATA3 Regulation of NCAPH Promoter
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The binding of GATA3 to the NCAPH promoter was examined using a ChIP assay with a kit (Beyotime Institute of Biotechnology) as per the manufacturer’s suggestions. SKOV3 cells were cross-linked by 1% formaldehyde for 15 min at room temperature, and the cell lysates in the lysis buffer were sonicated to achieve chromatin fragments. ChIP was conducted after incubation with anti-GATA3 antibody (Cell Signaling Technology, Danvers, MA, USA). The enrichment of indicated proteins in NCAPH promoter was evaluated by PCR.
6
Examining SPDEF-GALNT7 Promoter Binding
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ChIP assay was performed to detect the molecular interactions of SPDEF with the promoter of GALNT7 according to the manufacturer’s instruction (Beyotime, China). The primers of ChIP -qPCR listed in Table S2 .
7
Identifying Nr5a2-Nanog Promoter Interactions
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ChIP assay was performed to detect the molecular interactions of Nr5a2 with the promoter of Nanog according to manufacturer's instruction (Beyotime, China). Briefly, LLC‐SD cells were cross‐linked with 1% formaldehyde for 10 minutes at 37°C and cross‐linking was stopped by adding glycine solution for 5 minutes at room temperature. Subsequently, the lysed cells were isolated and sonicated on ice to shear DNA into fragments of 200 bp to 1 kb. Chromatin complexes were immunoprecipitated by incubating with anti‐NR5A2 or control anti‐IgG antibodies at 4°C overnight with rotation. The input DNA was isolated from the sonicated lysates before immunoprecipitation as a positive control. After washing and de‐crosslinking, purified DNA was detected by qPCR with primers listed in Table 3 .
8
Chromatin Immunoprecipitation Assay for TAZ
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After introduction of the TAZ overexpression plasmid and addition of LPS, a ChIP assay was performed using the corresponding kit (Beyotime). Briefly, cells were cross-linked with 1% formaldehyde, and chromatin from lysed nuclei was fragmented by sonication. Sheared DNA was precipitated with an antibody against TAZ or control immunoglobulin G (IgG), followed by incubation with protein A/G beads. Then, enriched chromatin DNA was purified and subjected to real-time PCR using the specific primer (Table 1 ).
9
Examining KLF9-SCD1 Interaction by ChIP Assay
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The binding between KLF9 and SCD1 was determined using a ChIP assay (Beyotime. Nantong, China) [29 (link)]. The cells were cross-linked with methanol and terminated with glycine, followed by lysis and sonication. Partial supernatant was as Input, while others were incubated with an anti-KLF9 antibody overnight. Following the incubation, beads were added to harvest the protein-DNA complex and 5 M sodium chloride (NaCl) was added to retrieve the DNA. The enrichment was determined using RT-qPCR as described above.
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