Ez magna chip a g assay kit

The chromatin immunoprecipitation (ChIP) assay was done on HT22 cells cultured in IL-6 (40ng/mL) to promote the STAT3 activation by SeqHealth (Wuhan, China). Following the above-mentioned processes, the cell protein-DNA complexes were cross-linked using 1% formalin in PBS on a rocking device at room temperature for 15min. The formaldehyde was quenched by adding 125mM glycine and rocked for 5min at room temperature. Afterward, the cells were collected and treated with lysis buffer in 500μL per 5×10⁶ cells on ice for 10min. The chromatin was sheared to an average length of about 1kb using sonication. After ultracentrifugation (10min) with a refrigerator at 12,000×g, we collected the supernatants and performed ChIP using the EZ-Magna ChIP A/G Assay kit (Millipore, 17-10086, United States). The chromatin mixture was immunoprecipitated with anti-phospho-STAT3 (Tyr705) antibody (1:100, Cell Signaling Technology, 9,145, United States). The DNA was purified and sequenced with ChIP-seq.

The assay was performed using EZ-Magna ChIP A/G assay kit (Millipore) according to the manufacturer's instructions with minor modifications. Briefly, HeLa cells were treated with 1 μg/ml staurosporine for 10 h, with 32 mM H₂O₂ for 4 h, or heated at 65 °C for 1 h and incubated for 3 h. The conditioned media were fixed with 1% formaldehyde for 10 min, quenched with 1X glycine for 5 min and concentrated. The concentrated media were incubated overnight with goat polyclonal anti-histone H1 antibody (Santa Cruz Biotechnology), goat polyclonal anti-HMGB1 antibody (Santa Cruz Biotechnology) or normal goat serum. The antibody–protein–DNA complexes were captured by incubation with EZ-Magna ChIP A/G. Genomic DNA segments from complexes were eluted, digested with proteinase K digestion and purified using spin columns. Aliquots of the DNA were used as a template for PCR amplification using 35 cycles of 55 °C annealing temperature. The GAPDH primers for PCR amplification were 5′-CCCCTTCATTGACCTCAACTAC-3′ and 5′-GAGTCCTTCCACGATACCAAAG-3′.

The EZ‐Magna ChIP A/G Assay Kit (Millipore) was used for chromatin immunoprecipitation (ChIP) assays according to the instruction manual from the manufacturer. Cells were treated with 1% formaldehyde for chromatin crosslinking and then collected and lysed with cell membrane extraction buffer to obtain the nuclei. Chromatin was then sheared by sonication into 200‐1000 bp DNA fragments and immunoprecipitated with normal rabbit IgG control or an anti‐hnRNP A1 antibody (Abcam). The precipitated DNA was then purified and went through PCR amplification for detection. ChIP primers designed for qPCR are listed in Table S6.

With the EZ-Magna ChIP A/G Assay Kit (Millipore, US), chromatin immunoprecipitation assays were performed following the manufacturer's protocol. The antibody against c-Myc was purchased from Abcam (US). Primers flanking the c-Myc binding sites on the miR-21 promoter were used for qPCR, and the sequences were as follows: forward, 5'-CATTGCACACCCTCTGGGGAAATT-3, and reverse 5'-CAAGACTTCATTCTAAATACACAG-3'.