MAGnify™ Chromatin Immunoprecipitation System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-The 1.25 M glycine must be at room temperature before use. -The Lysis Buffer must be at room temperature and fully resuspended before use. Vortex briefly to resuspend.	-For each ChIP reaction, use 10,000–300,000 cells or 0.167–5 mg of tissue. -1–10 μg of antibody is a typical starting range. - 10-minute crosslinking step using formaldehyde at a 1% final concentration. -Keep the cell lysate cooled on ice during sonication.

Upstream tips
-The 1.25 M glycine must be at room temperature before use. -The Lysis Buffer must be at room temperature and fully resuspended before use. Vortex briefly to resuspend.
Protocol tips
-For each ChIP reaction, use 10,000–300,000 cells or 0.167–5 mg of tissue. -1–10 μg of antibody is a typical starting range. - 10-minute crosslinking step using formaldehyde at a 1% final concentration. -Keep the cell lysate cooled on ice during sonication.

Publication protocol

ChIP assays were carried out using the ChIP Assay Kit (Thermo) as described previously [22]. The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and L4–L6 spinal dorsal horn were removed immediately and placed in 1% formaldehyde for 2 min. Then, DNA was fragmented by sonication and digested with micrococcal nuclease. After adding ChIP dilution buffer into DNA sample, 100 μl of the sample was saved as input. The precleared chromatin solution was incubated with anti-p-STAT3 antibody (Cell Signaling Technology, USA) or anti-acetyl-Histone H3 antibody (Abcam, UK) or anti-acetyl-Histone H4 antibody (Millipore, USA) overnight at 4 °C. The “IgG” immunoprecipitation was applied for the negative control. Next day, DNA was purified from the immunecomplexes and input fractions following the antibody/DNA complexes were captured, washed, eluted, and reverse cross-linked. The purified DNA was then resuspended in the nuclease-free water, and real-time quantitative PCR was performed on the sample as described in the above methods. Finally, the ratio of ChIP/input in the L4–L6 spinal dorsal horn was calculated. Primers 5′-CTCAATTCATGGTATTCTAAG-3′ and 5′-GAAGAAATTCATACATCACTG-3′ were designed to amplify a − 1148/− 1057 region relative to the transcription start site of rat Nalp1 promoter, containing the STAT3-binding site. Primers 5 -TCAAACACCCATTCACAGAGC-3 and 5 -CAAGACGCTCTTCTGTTGTTG-3 were designed to amplify a − 139/− 55 region relative to the transcription start site of mouse Nalp1 promoter, containing the STAT3-binding site.

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