- Table of Content
Learn the stepwise protocol for Histone chromatin immunoprecipitation (ChIP). It includes cell collection, DNA- Protein Cross-linking, cell lysis, chromatin shearing, and magnetic immunoprecipitation.
|Number of Cells||Trypsin-EDTA Volume|
|3 x 106||1 mL|
|1 x 107||3 mL|
|5 x 107||15 mL|
Note: Do not continue trypsin treatment longer than necessary as prolonged treatment with trypsin may damage the cells. Regularly check if the cells start to detach. Immediately add fresh culture medium to the cells when they are detached (refer to the table below for volume). This will inactivate trypsin. Transfer cell suspension to a 50 mL tube.
|Number of Cells||Culture Medium Volume|
|3 x 106||2 mL|
|1 x 107||6 mL|
|5 x 107||30 mL|
Before sequencing the samples, we recommend analyzing the immunoprecipitated DNA by qPCR using at least one positive and one negative control target. The kit contains a positive (H19 imprinting control region) and negative (Myoglobin exon 2) control primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade CTCF antibody) in SYBR Green qPCR assay using the protocol described below. Use your own method of choice for analyzing the appropriate control targets for your antibodies of interest.
Prepare the qPCR mix as follows (20 μL reaction volume using the provided control primer pairs):
This protocol has been optimized forchip-seq histone modificationl on an Illumina HiSeq sequencer. However, other sequencing systems such as the Illumina MiSeq or the Thermo Fisher SOLiD systems can also be used. Refer to their
sequencing protocols for the generation of sequencing data.