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

To validate that STAT3 binds the target sequences predicted by the bioinformatic analysis, we performed chromatin immunoprecipitation using a Pierce Agarose ChIP Kit (Thermo Fisher Scientific). Briefly, cells were cross-linked in 1% formaldehyde followed by sequential cell and nuclear membrane lysis. After the second lysis (nuclear lysis), sonication was performed to generate sheared DNA fragments. Supernatants of the final lysates were transferred to an immunoprecipitation system containing a spin column and rabbit anti-human STAT3 antibody (Cell Signaling Technology). The DNA–protein–STAT3 antibody complex was precipitated with protein A/G plus agarose for later elution. After elution, DNA was recovered and extracted for qPCR detection (primers specific to potential target sequences (listed in the Supplementary Materials) were identified by promoter deletion and bioinformatic analyses). Routine input control using 10% volume of the sonicated lysate was performed.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Pierce™ Agarose ChIP Kit below.

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