Pierce™ Agarose ChIP Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-If you are unfamiliar with cell type being used, culture an extra dish of cells for determining cell number. Before crosslinking, trypsinize and determine the cell number from the extra dish of cells.	-After incubation, it is essential to open the column cap before plug removal to equalize the pressure within the column. Failure to open the cap before plug removal will result in sample loss.	-If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use.

Upstream tips
-If you are unfamiliar with cell type being used, culture an extra dish of cells for determining cell number. Before crosslinking, trypsinize and determine the cell number from the extra dish of cells.
Protocol tips
-After incubation, it is essential to open the column cap before plug removal to equalize the pressure within the column. Failure to open the cap before plug removal will result in sample loss.
Downstream tips
-If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use.

Publication protocol

Chromatin Immunoprecipitation was performed by Pierce Agarose ChIP Kit (Pierce, Thermo Fisher
Scientific, Monza, Italy).Briefly, podocytes subcultured on T75 flask in triplicate, at a density of 2×106
cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature before adding glycine
solution to stop the reaction. Cell lysis was followed by Micrococcal nuclease digestion, 1U/μl for 15 minutes
at 37°C with production of 100- 400bp DNA fragments, as observed after 2% agarose gel electrophoresis
and a portion of total cross-linked lysate was saved for successive analysis (INPUT). Immunoprecipitation
was obtained by incubating overnight at 4°C the sheared chromatin with 10μg of NeuroD (G20) X antibody
for ChIP applications (SantaCruz), or with Normal rabbit IgG as negative control (Pierce), followed by
incubation with 20μl of ChIP Grade Protein A/G Agarose (Pierce) for 1 hour at 4°C on rocking platform.
The total nuclear unlinked extract (unlinked) was kept for Western Blot (WB) evaluation; the subsequent
IP elution and DNA recovery, collected for both WB and PCR analysis, followed standard instructions.
The concentrations of DNA from INPUT, and from ChIP were calculated after proteinase K treatment by
spectrophotometric determination.

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Manufacturer protocol

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