Although troubleshooting a ChIP-Seq experiment can be tricky at times, there are certain aspects of the ChIP procedure that should be of focus when trying to achieve optimal results. Focusing on these areas of the chromatin IP experiment can help a researcher set their ChIP-seq study up for success and, ultimately, publication.
From choosing the right antibody to shearing DNA, each step is vital to the success or failure of your chromatin immunoprecipitation experiment and should be completed with precision and accuracy. Follow these ChIP tips when preparing for a ChIP-seq study:
||Tips & RECOMMENDATIONS
- The antibody quality is one of the biggest causes of an unsuccessful ChIP experiment if it does not specifically enrich DNA-fragments bound by the target protein
- An antibody against the native protein is typically desired, but a tag antibody may also be used
- The ChIP antibody must be specific and sensitive enough. Testing your antibody with a Western Blot prior to the experiment is helpful, but this will not always be an accurate measurement of whether an antibody will perform well in ChIP. The only way to determine if an antibody will be successful for ChIP and ChIP-Seq is to complete a ChIP assay or use a commercially available ChIP Antibody Validation Kit.
- A polyclonal antibody will decrease the chance of masked epitopes during crosslinking, yet a monoclonal antibody has less variation between batches and recognizes a single epitope, increasing its sensitivity
- 1-10 ug of the antibody for each IP is a typical amount
- In general, for each immunoprecipitation performed, 25 μg of chromatin is recommended
- To avoid high background in non-specific antibody controls, a pre-clearing step is recommended. Pre-incubate the lysed sample with magnetic beads to “pre-clear” and reduce nonspecific immunoprecipitation
- Some suppliers’ A/G beads do not provide the cleanest results in the non-specific control, and it is crucial to buy A/G beads that will be highly efficient for your ChIP assay. It is advised to research products in advance and choose the optimal one for your assay.
- Desired fragment size is between 300 and 1000 bp
- To monitor shearing of the DNA fragments, use an agarose gel and adjust sonication accordingly
- Choose the appropriate sonication system to ensure fragments are consistent and uniform
- Illumina is the most popular high throughput sequencing platform for ChIP-seq
- Various platforms come with inherent biases – it is best to understand these biases and keep them in mind when interpreting ChIP-seq results