Chromatin immunoprecipitation and sequencing (ChIP-Seq) is a technique that allows us to analyze DNA-protein interactions at a genome-wide level.
In this technique, we first cross-link chromatin complexes, isolate them from the cell nuclei and fragment them. We can then purify chromatin fragments containing our protein of interest by immunoprecipitation. After this, the DNA fragments are purified and sequenced. We can use the sequencing results to determine the DNA regions our protein of interest interacts with.
Here we provide a detailed protocol and tips for performing cross-linking ChIP-seq method. At the end of this protocol, we will have prepared purified DNA ready for library preparation sequencing.
Our protocol is optimized for HeLa cells using chromatin from 1x107 cells per ChIP sample. The protocol also highlights the differences in the ChIP-seq procedure for various protein types, such as histones and transcription factors.
Before isolating the chromatin from your cell samples, we should prepare the ChIP-grade antibodies and beads so they’re ready for immunoprecipitation.
Prepare a slurry of protein A/G beads.
Beads can be vortexed briefly to help resuspend them before preparing the slurry.
Beads for multiple experiments can be prepared together in a pool.
Wash the beads twice with an excess of ice-cold PBS.
Block the beads with blocking buffer.
Wash the beads twice with 1 mL of RIPA-150.
Bind the beads to ChIP-grade antibodies.
If you pooled beads together previously, you could split them into individual samples at this step.
Refer to the manufacturer’s advice for the exact antibody concentration.
We use formaldehyde for cross-linking cells to preserve DNA-protein interactions. The cross-links will be removed later when ready for sequencing.
Wash cells and suspend in ~ 25 mL of ice-cold PBS.
We use 1x107 cells per sample. Ensure you grow enough cells for your experiment.
Cross-link cells in 1% formaldehyde.
Quench the cross-linking with glycine.
Wash cells twice in PBS.
When discarding the waste containing formaldehyde, please follow your local regulations for its disposal.
Detach adherent cells and place them in a fresh tube
Before we can proceed to fragment DNA, it is best to isolate the nuclei of cells to reduce cytoplasmic proteins.
Incubate cells in nuclear extraction buffer 1.
The first extraction buffer is a gentle buffer.
We use 1x107 cells per sample. You might need to optimize the buffer amount for the cell number used in your experiment.
Incubate cells in nuclear extraction buffer 2.
We use 1x107 cells per sample. You might need to optimize the buffer amount for the cell number used in your experiment.
At this stage, we must sonicate the cross-linked nuclear lysate to shear the DNA into fragments. The sonication step will require optimization depending on the cell line used and the protein being targeted. Please note that the following protocol is based on work in HeLa cells.
Histone targets can be sonicated more than non-histone targets as the nucleosomes are protected from fragmentation due to their close association with histone proteins. In contrast, non-histone targets can benefit from larger fragment sizes and less sonication.
Besides sonication, you may want to consider other forms of fragmentation, such as MNase treatment.
Spin down cells to a pellet and resuspend in the histone or non-histone sonication buffer, depending on your target of interest.
We use 1 x107 in 350 µL of sonication buffer for HeLa cells.
Sonicate lysate to shear DNA to an average fragment size of 150–300 bp for histone targets or 200–700 bp for non-histone targets.
This step will require optimization depending on the cell line used and the protein of interest.
Pellet cell debris using a centrifuge at 17,000 g at 15 mins at 4°C.
Note that such high centrifugation speed is needed to completely separate chromatin from the nuclear pellet.
Chromatin can be flash frozen at this point and stored at -80 C for up to 2 months.
Before immunoprecipitating the target protein, we should elute a small test sample. This will allow us to determine the size of the DNA fragments and check they’ve not been degraded.
For histones, we should aim for an average fragment size of 150 – 300 bp; for non-histones, it’s 200 – 700 bp.
Remove a 50 µL aliquot of sonicated sample for testing.
Incubate the sonicated sample with the direct elution buffer.
Incubate the sample with RNase A solution.
RNase A is included in the buffer as high levels of RNA will interfere with DNA purification when using a PCR purification kit.
Incubate the sample with Proteinase K solution.
Proteinase K cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. Cross-links between proteins and DNA are disrupted, which aids DNA purification.
Purify the DNA using your standard laboratory procedures.
Run the DNA on a 1-2% agarose gel to determine fragment size.
If the fragment size is too low or we have lots of small fragments, the sample has been degraded. In this case, we’ll need to repeat the protocol from the start.
If the fragment size is too large, sonication has not sheared DNA enough. In this case, we’ll need to repeat Stage 4 (Sonication) on your existing sample.
Pellet cell debris from sonicated sample prepared in Stage 4, assuming DNA fragment size is validated.
This supernatant contains the chromatin that will be used for the following stages.
We’re now ready to perform immunoprecipitation by mixing the sonicated sample with antibody-bound magnetic beads. This will purify DNA fragments associated with the protein of interest.
Prepare the amount of IP buffer required for your number of samples.
Depending on your target of interest, you’ll need to prepare the IP buffer for histone or non-histone targets.
Thaw chromatin samples (if frozen) and dilute them ~ 1/10 in the IP buffer.
In this protocol, we calibrate the 100 µL sample to have the equivalent chromatin of 1x107 cells. If you have pooled your samples together, you can split them into separate tubes at this step.
Incubate the chromatin samples with antibody-coupled beads overnight at 4 °C with gentle rotation.
At this stage, the antibodies will bind to the chromatin.
Wash the beads with a sequence of buffers to remove background.
The beads are washed with three buffers; the stringency of the wash increases with each change of buffer.
At this stage, we elute and purify the DNA to prepare it for sequencing.
Incubate the sonicated sample with the direct elution buffer.
Incubate the sample with RNase A solution.
RNase A is included in the buffer as high levels of RNA will interfere with DNA purification when using a PCR purification kit.
Incubate the sample with Proteinase K solution.
Proteinase K cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. Cross-links between proteins and DNA are disrupted, which aids DNA purification.
Purify the DNA using your standard laboratory procedures.
Proceed to DNA library preparation and sequencing.
You may wish to run quantitative PCR before sequencing to check the quality of the DNA fragments.