Pierce agarose chip kit

CHIP assay was performed using a Pierce^TM Agarose CHIP Kit (Thermo Scientific, Rockford, IL, USA). Briefly, L02 cells were treated with or without Hyp (200 μM) for 24 h. DNA and proteins were cross-linked by incubating cells with 1% formaldehyde for 10 min at room temperature. Excess formaldehyde was quenched with glycine for five minutes. Cells were then lysed and nuclei were digested using micrococcal nuclease. Sheared chromatin was diluted and immunoprecipitated with 10 μg of anti-Nrf2, anti-Bach1 or control IgG antibody. DNA-protein complexes were eluted from the protein A/G agarose beads using a spin column and were reverse cross-linked by incubating with NaCl at 65°C.

The ability of RUNX1 to enrich the LRRC15 promoter was assayed using a Pierce^TM Agarose ChIP kit (Thermo Fisher Scientific). Briefly, RA-FLS were cross-linked with 1% paraformaldehyde at room temperature for 10 min, then 0.125 M glycine was added to arrest the crosslinking reaction. The cells were centrifuged, lysed with a buffer containing Protease Inhibitor Cocktail, and centrifuged again to obtain nuclei. An ultrasonic disruptor was set to 4 s per ultrasonic cycle at 8-s intervals for 6 min. After addition of elution buffer containing RNase A, the samples were incubated with Proteinase K for 2 h at 62°C. Fragmented chromatin extracts were incubated with ChIP Grade RUNX1 antibody (1:50, ab272456; Abcam) or normal rabbit IgG (Thermo Fisher Scientific) overnight at 4°C, and with protein A/G magnetic beads for 2 h at 4°C. After thorough washing, elution, and de-crosslinking, purified DNA was used for qPCR analysis. PCR primer sequences for the LRRC15 promoter were as follows: forward primer: 5′-TGCCTACTTACCAGCAGCTC-3′, reverse primer: 5′-GCCAGCTATGTGTACCCTGT-3′.