ChIP-qPCR Protocol for Hypoxia Response

ChIP-qPCR was performed according to previous protocols with slight modifications (Hu et al., 2020 (link)). Briefly, 1 × 10⁶ hMSCs pretreated with 3% O₂ for 48 h were crosslinked by 1% (v/v) formaldehyde diluted in PBS for 13 min. The reaction was stopped by an incubation in 0.125 mol/L Glycine for 5 min at room temperature. After washes with PBS, cells were resuspended in ice-cold lysis buffer (50 mmol/L Tris-HCl, 10 mmol/L EDTA, 1% SDS, pH 8.0) for 5 min. After sonication by a Bioruptor® Plus device (Diagenode), supernatants were incubated overnight at 4°C with Protein A/G dynabeads (Thermo Fisher Scientific, 10004D) conjugated with anti-HIF-1α, or normal rabbit IgG. Subsequently, elution and reverse cross-linking were performed at 68°C for 3 h on a thermomixer. DNA was then isolated by the phenol–chloroform–isoamylalcohol extraction and ethanol precipitation method, and the purified DNA was used for qPCR detection. Primers used in this study are listed in Table S3.