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 analyses were performed on chromatin extracts using MAGnify Chromatin Immunoprecipitation System kit (Invitrogen), according to manufacturer's specifications. Cell cultures (about 1×106 cells/ml) were cross-linked, in standard culture dishes, at room temperature for 10 min by formaldehyde 37% (final concentration 1%). Reaction was stopped by 5 min incubation in 0.125 M Glycine. Cell monolayer was harvested by scraping in ice-cold PBS containing protease inhibitors. After cell lysis (final concentration of cell: 106 cells/50 μl) chromatin was sonicated using Bioruptor NextGen (Diagenode) to High Power, 18 cycles for 30 seconds ON, 30 seconds OFF. Average size of sonicated DNA was around 400 bp, as measured by agarose gel electrophoresis. Aliquots containing 200.000 cells were snap-freezed and stored at -80°C. Sheared chromatin was immunoprecipitated with anti-acetyl-Histone H3 or anti-acetyl-Histone H4, or rabbit IgG as negative control. DNA amplification was performed using SsoAdvanced SYBR Green supermix on a MiniOpticon Real-time PCR System (Bio-Rad).

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Manufacturer protocol

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