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Cross-linking ChIP-Seq (X-ChIP-Seq) protocol

Detailed procedure and tips for cross-linking ChIP using ChIP-seq and ChIP-qPCR methods.
Last edited Tue 31 Jan 2023

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. 

Stage 1 - Bead preparation

Before isolating the chromatin from your cell samples, we should prepare the ChIP-grade antibodies and beads so they’re ready for immunoprecipitation.

Materials required

  • ChIP-grade primary antibody
  • Protein A magnetic beads 
  • Protein G magnetic beads
  • Ice-cold PBS
  • RIPA-150 (50 mM Tris-HCl pH=8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate)
  • Blocking buffer (0.5% w/v BSA, 1x Protease inhibitor made up in RIPA-150 buffer) 

Steps

1

Prepare a slurry of protein A/G beads.

  • Add 12.5 µL of protein A beads and 12.5 µL of protein G beads for each sample you wish to run to a tube to prepare a 50:50 mix.
2

Wash the beads twice with an excess of ice-cold PBS.

  • Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
  • Aspirate and discard the solution, keeping the beads in the tube.
  • Add an excess of fresh, ice-cold PBS to wash the beads.
  • Collect the beads using the magnet and aspirate and discard the solution.
  • Repeat the PBS wash step once more so that the beads have been washed twice in total.
3

Block the beads with blocking buffer.

  • Remove the PBS solution from the last step.
  • Add 1 mL of blocking buffer.
  • Incubate beads in blocking buffer for 30 mins at 4°C with gentle rotation.
4

Wash the beads twice with 1 mL of RIPA-150.

  • Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
  • Aspirate and discard the solution, keeping the beads in the tube.
  • Add ~ 1 mL of fresh RIPA-150 and repeat the steps above.
  • Repeat the RIPA-150 wash step once more so that the beads have been washed twice in total.
5

Bind the beads to ChIP-grade antibodies.

  • Resuspend the beads in RIPA-150 buffer so that each experiment has 25 µL of bead slurry in 500 µL of RIPA-150 in a separate tube.
  • Add your antibody to the resuspended bead mix. We use 4 µg for histone targets and 8 µg for non-histone targets.   
  • Incubate for ~6 hrs or overnight at 4°C with gentle rotation.

Stage 2 - Harvesting and cross-linking cells

We use formaldehyde for cross-linking cells to preserve DNA-protein interactions. The cross-links will be removed later when ready for sequencing.

Materials required

  • Cells in culture
  • Ice-cold PBS
  • Cross-linking agent (formaldehyde)
  • Quencher (glycine)
  • Cell scraper 

Steps

1

Wash cells and suspend in ~ 25 mL of ice-cold PBS.

  • Remove cells from the incubator at ~90% confluence.
  • Gently rinse cells twice with 10-20 mL of ice-cold PBS.
  • Discard the liquid and pipette in ~27 mL of ice-cold PBS.
2

Cross-link cells in 1% formaldehyde.

  • Gently add formaldehyde to the flask to a final concentration of 1%.
  • Incubate cells for 10 min at room temperature with gentle swirling.
3

Quench the cross-linking with glycine.

  • Gently add glycine to the flask to a final concentration of 125 mM.
  • Incubate cells for 5 min at room temperature with gentle agitation.
4

Wash cells twice in PBS.

  • Discard the liquid in the flask.
  • Gently rinse cells with 10 mL of PBS.
  • Discard liquid after each wash, adding fresh PBS after each wash.
5

Detach adherent cells and place them in a fresh tube

  • Discard the liquid in the flask and place the flask on ice.
  • Add ~ 5 mL of PBS.
  • Scrape cells thoroughly to detach them from the bottom of the flask.
  • Pour the suspended cells into a fresh tube.
  • Repeat as necessary.

Stage 3 - Isolating nuclear fraction

Before we can proceed to fragment DNA, it is best to isolate the nuclei of cells to reduce cytoplasmic proteins.

Materials required

  • Nuclear extraction buffer 1 (50 mM HEPES-NaOH pH=7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% TritonX-100, 1x protease inhibitors)
  • Nuclear extraction buffer 2 (10 mM Tris-HCl pH=8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x protease inhibitors)
  • Centrifuge

Steps

1

Incubate cells in nuclear extraction buffer 1.

  • Spin cells down to a pellet (1,500 x g, 5 mins, 4°C).
  • Resuspend the pellet in ~ 2 mL of extraction buffer 1.
  • Incubate in the buffer for 15 min at 4°C with rocking.
2

Incubate cells in nuclear extraction buffer 2.

  • Spin cells down to a pellet (1,500 x g, 5 mins, 4°C).
  • Resuspend the pellet in ~ 2 mL of extraction buffer 2.
  • Incubate in the buffer for 15 min at 4°C with rocking.

Stage 4 - Sonication

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.

Materials required

  • Histone sonication buffer (50 mM Tris-HCl pH=8.0, 10 mM EDTA, 1 % SDS, protease inhibitors) for histone targets
  • Non-Histone sonication buffer (10 mM Tris-HCl pH=8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 % sodium deoxycholate, 0.5 % sodium lauroylsarcosine, protease inhibitors) for non-histone targets
  • Sonicator
  • Centrifuge

Steps

1

Spin down cells to a pellet and resuspend in the histone or non-histone sonication buffer, depending on your target of interest.

  • Spin cells down to a pellet at 1,500 g for 5 mins, 4°C, and discard the supernatant.
  • Resuspend the pellet in the sonication buffer. 
2

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.

3

Pellet cell debris using a centrifuge at 17,000 g at 15 mins at 4°C.

  • Discard the pellet.
  • Keep the supernatant and transfer it into a new tube. 

Stage 5 - Determination of DNA fragment size

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.

Materials required

  • Direct elution buffer (100 mM NaHCO3, 1 % SDS)
  • RNAse (10 mg/mL solution)
  • Proteinase K (20 mg/mL solution)
  • Agarose gel (1 – 2 %)
  • DNA molecular weight marker (100 bp)

Steps

1

Remove a 50 µL aliquot of sonicated sample for testing.

2

Incubate the sonicated sample with the direct elution buffer.

  • Shake samples in a thermomixer at 65 °C for 4.5 h or overnight.
3

Incubate the sample with RNase A solution.

  • Add 2 µL of RNase A solution and heat at 37 °C for 30 min.
4

Incubate the sample with Proteinase K solution.

  • Add 2 µL of Proteinase K solution and heat at 55 °C for 1 h.
5

Purify the DNA using your standard laboratory procedures.

  • Commercial PCR kits or phenol:chloroform extraction.
6

Run the DNA on a 1-2% agarose gel to determine fragment size.

  • Compare sample bands observed with a marker that has fragments of known length.
7

Pellet cell debris from sonicated sample prepared in Stage 4, assuming DNA fragment size is validated.

  • Spin down cells at 17,000 g for 15 min at 4 °C.
  • Discard the pellet.
  • Keep the supernatant and transfer it into a new tube. 
  • Store at – 20 °C until ready for use.

Stage 6 - Immunoprecipitation

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.

Materials required

  • Histone Target IP buffer (50 mM Tris-HCl pH=8.0, 167 mM NaCl, 1.10 % Triton X-100, 0.11 % Sodium deoxycholate)
  • Non-histone Target IP buffer (10 mM Tris-HCl pH=8.0, 100 mM NaCl, 1 mM EDTA pH=8.0, 0.5 mM EGTA pH=8.0, 0.1% sodium deoxycholate, 0.5% sodium lauroylsarcosine, 1% TritonX, 1x protease inhibitors)
  • Protease inhibitor cocktail (example ab65621)
  • Antibody-coupled magnetic beads (prepared in step 1.1)
  • RIPA-150 (50 mM Tris-HCl pH=8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate)
  • RIPA-500 (50 mM Tris-HCl pH=8.0, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate)
  • RIPA-LiCl (50 mM Tris-HCl pH=8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate)
  • TE Buffer (10 mM Tris-HCl pH=8.0, 1 mM EDTA)
  • Magnetic rack
  • Tube rotator

Steps

1

Prepare the amount of IP buffer required for your number of samples.

  • Add protease inhibitors just before use.
2

Thaw chromatin samples (if frozen) and dilute them ~ 1/10 in the IP buffer.

  • Add 100 µL of sample to ~900 µL of IP buffer.
3

Incubate the chromatin samples with antibody-coupled beads overnight at 4 °C with gentle rotation.

4

Wash the beads with a sequence of buffers to remove background.

  • Add 1 mL of RIPA-150 buffer and place the tube on a rocker for 2 min.
  • Place the tube on a magnetic rack for ~1 min; the beads should be pulled by the magnet to the bottom of the tube.
  • Aspirate and discard the solution, keeping the beads in the tube.
  • Repeat RIPA-150 wash steps to wash twice in total.
  • Mix the beads with ~1 mL of the RIPA-500 wash buffer for 2 min on a rocker. 
  • Place the tube on a magnetic rack for ~1 min; aspirate and discard the solution, keeping the beads in the tube.
  • Repeat the RIPA-500 wash steps to wash twice in total.
  • Mix the beads with ~1 mL of the RIPA-LiCl wash buffer for 2 min on a rocker. 
  • Place the tube on a magnetic rack for ~1 min.
  • Aspirate and discard the solution, keeping the beads in the tube.
  • Mix the beads with ~1 mL of the TE Buffer for 1 min on a rocker. 
  • Place the tube on a magnetic rack for ~1 min; aspirate and discard the solution, keeping the beads in the tube.

Stage 7 - Elution and reversal of cross-links

At this stage, we elute and purify the DNA to prepare it for sequencing.

Materials required

  • Elution buffer (100 mM NaHCO3, 1 % SDS)
  • Magnetic rack
  • PCR kit

Steps

1

Incubate the sonicated sample with the direct elution buffer.

  • Shake samples in a thermomixer at 65 °C for 4.5 h or overnight.
2

Incubate the sample with RNase A solution.

  • Add 2 µL of RNase A solution and heat at 37 °C for 30 min.
3

Incubate the sample with Proteinase K solution.

  • Add 2 µL of Proteinase K solution and heat at 55 °C for 1 h.
4

Purify the DNA using your standard laboratory procedures.

  • Use commercial PCR kits or phenol:chloroform extraction.
5

Proceed to DNA library preparation and sequencing.

  • It is easier to use a highly sensitive commercial kit to determine the concentration of the immunoprecipitated DNA.