ChIP-seq Kit Troubleshooting Guide

The following guide serves as a checklist for the notes/possible issues and solutions/comments with respect to the critical steps in the ChIP-seq kit assays.

Cross Linking

Note/Possible Issue

Solution/Comment

1

Cross-linking is too weak or too strong.

Make sure the fixation step is performed for the correct period of time, at the right temperature and with the correct formaldehyde concentration [e.g., incubate for 10 to 20 min at room temperature with 1% formaldehyde final concentration (weight/volume)]. Also, use high quality, fresh formaldehyde.

2

Proteins have unique ways of interacting with the DNA. Some proteins are not directly bound to the DNA but interact with other DNA-associated proteins.

Very short or very long cross-linking time can lead to DNA loss and/or elevated background. Thus, the optimal cross-linking time should be found empirically as maximal specificity and efficiency of ChIP.

3

Both cross-linking time and formaldehyde concentration are critical.

Cross-linking can affect both efficiency of chromatin shearing and efficiency of specific antigen immunoprecipitation. Shorter cross-linking times (5 to 10 min) and/or lower formaldehyde concentrations (<1%, weight/volume) may improve shearing efficiency while, for some proteins especially those that do not directly bind DNA, this might reduce the efficiency of cross-linking and thus the yield of precipitated chromatin.

4

The optimal duration of cross-linking varies between cell type and protein of interest.

It is possible to optimize the fixation step by testing different incubation times: such as 10, 20 and 30 min. Do not cross-link for longer than 30 min as cross-links of more than 30 min cannot be efficiently sheared.

5

Efficient fixation of a protein to chromatin in vivo is a crucial step for ChIP. The extent of cross-linking is probably the most important parameter.

Two major problems concerning the subsequent immunoprecipitation step should be taken into account: 1) An excess of cross-linking can result in the loss of material or reduced antigen availability in chromatin, or both. 2) The relative sensitivity of the antigen epitopes to formaldehyde. It is essential to perform the cross-linking step with care.

6

It is essential to quench the formaldehyde.

Use glycine to stop the fixation: quench formaldehyde with 125 mM glycine for 5 min at room temperature (add 1/10 volume of 1.25M glycine). Alternatively, wash the fixed cells properly and make sure you remove all of the formaldehyde.

Cell Lysis

Note/Possible Issue

Solution/Comment

1

Temperature is critical.

Perform cell lysis at 4°C (cold room) or on ice. Keep the samples ice-cold at all times during the cell lysis and use ice-cold buffers.

2

Protein degradation during lysis can occur.

Add the protease inhibitors to the lysis buffer immediately before use.

Cell Type

Note/Possible Issue

Solution/Comment

1

Kit protocol validation

HeLa, NCCIT 293T, Chondrocytes, P19, ASC (adipose stem cells) and Kerationocytes have been used to validate the magnetic ChIP protocol.

Chromatin Shearing

Note/Possible Issue

Solution/Comment

1

Optimal shearing conditions are important for ChIP efficiency.

Shearing conditions for each cell type must be optimized from cell collection, fixation to shearing method (settings of the sonicator apparatus).

2

Critical points for shearing optimization

1) Not to start with a too high concentration of cells (≤ 15 x 10e6 cells/mL is acceptable). 2) Keep samples cold (4°C).

Sheared Chromatin Analysis

Note/Possible Issue

Solution/Comment

1

Purify the DNA from the sheared chromatin as described in the kit protocol to analyze the shearing.

Extract total DNA from an aliquot of sheared chromatin and run on 1% agarose gel (stain with EtBr). In order to analyze the sheared chromatin on gel, take DNA purified from the sheared chromatin input. Some unsheared chromatin can be analyzed on gel as well. Chromatin equivalent to 100,000 cells, 1 million cells or more can for sure be visualized on a gel.

2

Do not load too much DNA on a gel.

Loading of large quantities of DNA on agarose gel can lead to poor quality pictures, which do not reflect the real DNA fragmentation. The DNA amount to load depends on the size of the well and on the gel size.

3

Agarose concentration

Do not use more than 1-1.5% agarose gel and run slowly (Volt/cm and time depend on the gel size).

4

Running buffer concentration

1X TAE or TBE is preferred to 0.5X TAE, which can lead to smears on agarose gel.

Sheared Chromatin Amount

Note/Possible Issue

Solution/Comment

1

Amount of sheared chromatin to be prepared

Most of the sheared chromatin is to be used in the ChIP experiment, but remember that some of the sheared chromatin is needed as control as it corresponds to the input sample for the ChIP experiment and it can also be checked on agarose gel.

Antibody Binding Beads

Note/Possible Issue

Solution/Comment

1

Beads are in suspension

The provided beads are coated with protein A. Resuspend into a uniform suspension before each use.

2

Bead centrifugation

Don’t spin the beads at high speed. Use gentle centrifugation (500 x g for 2-3 minutes). It is possible to centrifuge the 1.5 mL tubes at 1,000 – 2,000 x g for 20 sec.

3

Bead storage

Store at 4°C. Do not freeze.

4

Antibody binding capacity

Approximately 10 µg antibody per 30 µL beads

Protease Inhibitors

Note/Possible Issue

Solution/Comment

1

Storage

Some inhibitors are unstable in solution. The provided protease inhibitor mix should be kept frozen at -20°C, and thawed before use.

Other Enzyme Inhibitors

Note/Possible Issue

Solution/Comment

1

Specific enzyme inhibitors are not included in the kit such as phosphatase inhibitors.

Add phosphatase inhibitors or others to Buffers A and B, if necessary, depending on your research field and protein(s) of interest to be chromatin immune-precipitated. Add NaBu for histone ChIPs.

Negative ChIP Control(s)

Note/Possible Issue

Solution/Comment

1

Use non-immune IgG in the IP incubation mix.

Use the non-immune IgG fraction from the same species the antibodies were produced in.

2

Do not add antibody to the IP.

Incubation with beads, which were not coated with antibodies could also be used as a negative ChIP control.

3

Use a specifically blocked antibody in parallel.

Use one antibody in ChIP and the same antibody that is blocked with specific peptide. To specifically block an antibody: pre-incubate the antibody with saturating amounts of its epitope specific peptide for about 30 min at room temperature before use in the IP incubation mix. Use the blocked antibody as a negative control in parallel with the unblocked antibody.

Antibody in IP

Note/Possible Issue

Solution/Comment

1

Number of necessary negative controls

If multiple antibodies of the same species are to be used with the same chromatin preparation, then a single negative ChIP control is sufficient for all of the antibodies used.

2

Antibody is not working in ChIP.

Antibody-antigen recognition can be significantly affected by the cross-linking step resulting in loss of epitope accessibility and/or recognition.

3

Type of antibody used in ChIP

Use ChIP-grade antibodies. If not available, it is recommended to use several antibodies directed against different epitopes of the same protein. Verify that the antibodies can work directly in IP on fresh cell extracts. Also, when testing new antibodies, include known ChIP-grade antibodies as positive control for your ChIP assay.

4

Choosing the right antibody for ChIP

Be aware of the possible cross-reactivity of antibodies. Verify by Western blot analysis for the antibody specificity. Antigen affinity purification can be used to increase titer and specificity of polyclonal antibodies.

5

Binding of antibodies to protein A or protein G

There is a significant difference in affinity of different types of immunoglobulins to protein A or G. Therefore, in function of the antibody used for your ChIP, it is recommended to choose either protein A or protein G coated beads.

Species Immunoglobin Isotype Protein A Protein G
Human IgG1 +++ +++
IgG2 +++ +++
IgG3 - +++
IgG4 +++ +++
IgGM Use anti-human IgM
IgGF - +
IgGA - +
Mouse IgG1 + +++
IgG2a +++ +++
IgG2b ++ ++
IgG3 + +
IgGM Use anti-mouse IgM
Rat IgG1 - +
IgG2a - +++
IgG2b - ++
IgG2c + ++
Chicken All Isotypes - ++
Cow All Isotypes ++ +++
Goat All Isotypes - ++
Guinea Pig All Isotypes +++ ++
Hamster All Isotypes + ++
Horse All Isotypes ++ +++
Pig All Isotypes + ++
Rabbit All Isotypes +++ ++
Sheep All Isotypes - ++

Immuno-Selection Incubation

Note/Possible Issue

Solution/Comment

1

Best incubation time for immune-selection using the ultrasonic water bath

To incubate the sheared chromatin with antibodies for 15 to 30 min works for many antibodies, however, the kinetics for reaching equilibrium of epitope-antibody binding may differ for each antibody and target. Optimization might improve the results (e.g. the incubation time may need to be increased for some antibodies).

2

Principle for immuno-selection using the ultrasonic water bath work

The rate-limiting step in many immunoassays is associated with the slow kinetics of binding of macro-molecular antigen to antibody. It was demonstrated that the use of ultrasonic energy to enhance mass transport across liquid/solid interfaces can dramatically accelerate antigen binding to antibodies.

3

Water bath specifications

Model MT-3510. Capacity: 5.5 liters. Size (LxWxH): 29 x 15 x 15 cm. Frequency: 42 kHz. Max power requirement: 130 W. RF-Power: 130 W.

4

Use of kit without an ultrasonic water bath

This is fine but a long incubation at 4ºC should be used. Depending on the antibody and target to be chromatin immuno-precipitated, the times of incubation range from 2 to 16 hours and should be determined empirically for each antibody.

PCR

Note/Possible Issue

Solution/Comment

1

Primer design

Primer length: 18 to 24 nucleotides

Primer Tm: 60°C (+/- 3.0ºC)

% GC: 50% (+/- 4%)

2

Positive and negative controls

Negative PCR controls: PCR with DNA from samples immuno-precipitated with non-immune antibodies (negative IgG). Alternatively, PCR using DNA from ChIP samples and primers specific for a DNA region to which, your antigen of interest is not binding. Positive PCR control: PCR using input DNA.

3

No PCR signal

Include a positive PCR control as a control for your PCR mix (your primers, dNTP and Master Mix) using the input DNA or a DNA sample of the same origin.

4

High Ct values

Use more input chromatin

5

CtNegCtl and CtTarget [i.e., Mean threshold cycles of PCR done in duplicate on DNA samples from negative control ChIP (using non-immune IgG) and targeted ChIP (specific antibody)]

The ratio between target IP and negative control IP depends on the antibody used.

6

High background

Verify the following steps: Keep the antibody binding beads and DNA purifying slurry in suspension while adding to tubes. Check by eye that equal pellets of beads and slurry are present in each tube. Washes are critical.

7

Using end-point PCR rather than quantitative PCR

If gel electrophoresis is used to estimate intensities of PCR products, then relative occupancy of a factor at a locus is the ratio of the intensity of the target IP band to the negative control IP band.

Freezing

Note/Possible Issue

Solution/Comment

1

Samples can be frozen at several steps of the protocol.

Pellets of formaldehyde fixed cells can be stored at - 80°C for at least a year. Sheared chromatin can be stored at - 80ºC for months, depending on the protein of interest to be chromatin immuno-precipitated. Purified DNA from ChIP and input samples can be stored at -20ºC for months.

2

Avoid multiple freeze/thaw.

Snap freeze and thaw on ice (e.g. fixed cell pellets and sheared chromatin)