Dual cross-linking ChIP protocol (dual-X-ChIP)

Chromatin immunoprecipitation (ChIP) is a widely used method for investigating protein–DNA interactions. It allows you to determine whether a protein of interest binds to specific genomic regions or to map its distribution across the genome (ChIPseq).

This protocol describes how to perform ChIP using a dual crosslinking approach to improve recovery of transcription factors and chromatin-associated proteins. Dual crosslinking is particularly useful when working with proteins that have weak, transient, or indirect interactions with DNA.

In this method, EGS (ethylene glycol bis(succinimidyl succinate)) is used first to stabilize protein–protein interactions. EGS is a membrane-permeable, aminereactive crosslinker with a longer spacer arm than formaldehyde, enabling it to capture proteins that sit farther from the DNA or are part of larger multiprotein complexes. Formaldehyde is then added to crosslink these stabilized complexes to DNA.

Together, these steps help preserve fragile or dynamic transcriptional complexes, improving ChIP efficiency and increasing the likelihood of detecting proteins that do not directly contact DNA. As with any crosslinking method, optimization is important, as excessive fixation may reduce epitope accessibility or impact chromatin fragmentation.

Solutions

RIPA buffer

• 50 mM Tris-HCl pH8 150 mM NaCl
• 2 mM EDTA pH8 1% NP-40
• 0.5% Sodium Deoxycholate
• 0.1% SDS
• Protease Inhibitors (add fresh each time)

Wash buffer

• 0.1% SDS
• 1% Triton X-100 or 1% NP-40
• 2 mM EDTA
• 20 mM Tris-HCl pH 8.0
• 150 mM NaCl

TE buffer

• 10 mM Tris pH 8.0
• 1 mM EDTA

Elution buffer

• 1% SDS
• 100mM NaHCO₃

Stage 1 - Cross-linking and cell harvesting

Both formaldehyde and EGS (ethylene glycol bis (succinimidyl succinate)) are used in this protocol to dual cross-link the proteins to the DNA. Dual cross-linking is particularly effective for capturing transcription factors and chromatin-associated proteins with transient or indirect DNA interactions.

However, over-crosslinking can significantly impair downstream steps by reducing antigen accessibility, masking epitopes, and decreasing sonication efficiency. Therefore, careful optimization of both EGS concentration and cross-linking duration is essential.

This protocol is optimized for HeLa cells, and the following conditions serve as a recommended starting point:

• EGS: 1.5–2 mM final concentration
• Incubation: 20–30 minutes at room temperature
• Formaldehyde: 1% final concentration
• Incubation: 10 minutes at room temperature

All cross-linking steps should be performed in a certified fume hood with appropriate personal protective equipment (PPE) due to the hazardous nature of formaldehyde.

Steps

Start with two confluent dishes.

• The two dishes should be 150 mm² with 1x10⁷–5x10⁷ cells per dish.
• Cross-link proteins to DNA by adding 20 mL ice-cold PBS to each flask with EGS (300 mM) to a final concentration of 1.5 mM.
• Swirl gently at room temperature for 30 min in total, adding the formaldehyde when you have 10 min remaining: see step 2.

Add formaldehyde (37%) to each flask to final dilution.

• Final % should be 1% for transcription factor projects.
• Swirl gently at room temperature for 10 min (this timing will need to be optimized for different cell types).

Add 1.5 mL of 2.5 M glycine (125 mM final) to the media and incubate with shaking for 5 mins to quench formaldehyde.

Rinse cells twice with 10 mL cold PBS.

Add 5 mL of cold PBS, scrape dishes thoroughly with a cell scraper, and transfer into 50 mL tube.

Add 3 mL PBS to dishes, scrape again, and transfer the remainder of the cells to the 50 mL tube.

Centrifuge for 5 min at 4°C at 1,000 x g.

Carefully aspirate off supernatant.

• Resuspend the pellet in ChIP Lysis Buffer (750 μL per 1x10⁷ cells) and incubate for 10 mins on ice.

Nuclei preparation

To the pellet add 6ml Buffer I (50mM HEPES-NaOH pH=7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40,  0.25% TritonX-100, 1×protease inhibitors) to resuspend cells.

• Incubate at 4 °C for 10 min. 1,000 rpm 5 min centrifuge.

Collect pellets and resuspend in 6ml Buffer II (200mM NaCl, 1mM EDTA pH=8.0, 0.5 mM EDTA pH=8.0,  10mM Tris-Cl pH=8.0, 1× protease inhibitors), incubate at room temperature for 10min, then centrifuge at 1,000rpm for 5min.

Resuspend pellet nuclei in 200 ul Buffer III (50mM Tris-HCl pH=8.0, 10mM EDTA pH=8.0, 1% SDS 1× protease inhibitors).

Stage 2 - Sonication

Steps

Sonicate lysate to shear DNA to an average fragment size of 200–1,000 bp.

• The fragment size should be analyzed on a 1.5% agarose gel.

After sonication, pellet cell debris by centrifugation for 10 min at 4°C at 8,000 x g.

• Transfer supernatant to a new tube.

Remove 50 μL of each sonicated sample, to determine DNA concentration and fragment size.

Stage 3 - Determination of DNA concentration and 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

Remove a 50 µL aliquot of sonicated sample for testing.

Incubate the sonicated sample with the direct elution buffer.

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

Incubate the sample with RNase A solution.

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

Incubate the sample with Proteinase K solution.

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

Purify the DNA using your standard laboratory procedures.

• Commercial PCR kits or phenol:chloroform extraction.

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.

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

• Spin down cells at 17,000 x 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 4 - Immunoprecipitation

Steps

Using the chromatin prepared, dilute each sample 1:10 with RIPA Buffer.

• You will need one sample for the specific antibody and one sample for the control (beads only).
• Remove 50 μL of chromatin to serve as your input sample and store it at -20°C until further use.

Add primary antibody to all samples except the beads-only control and rotate at 4°C for 1 hour.

• The amount of antibody to be added should be determined empirically; 1-10 μg of antibody per 25 μg of DNA often works well.
• Protein A beads, protein G beads or a mix of both should be used. Table 1 shows the affinity of protein A and G beads to different immunoglobulin isotypes.

Table 1. The affinity of Protein A and G beads to different immunoglobin isotypes.

Preparation of protein A/G beads.

• If using both Protein A and Protein G beads, mix an equal volume of Protein A and Protein G beads and wash three times in RIPA buffer.
• Aspirate RIPA buffer and add herring sperm DNA to a final concentration of 75 ng/μL beads and BSA to a final concentration of 0.1 μg/μL beads.
• Add RIPA buffer to twice the bead volume and incubate for 30 min with rotation at RT.
• Wash once with RIPA buffer and add RIPA buffer to twice the bead volume.

Add 60 μL of blocked protein A/G beads to all samples and IP overnight with rotation at 4°C.

Centrifuge the immunoprecipitated samples for 1 min at 2,000 x g and remove the supernatant.

Wash three times in wash buffer.

• After each wash, centrifuge for 1 min at 2,000 x g and remove the supernatant.

Stage 5 - Elution and reverse cross-links

Steps

Elute DNA by adding 120 μL of elution buffer to the protein A/G beads.

• Vortex slowly for 15 min at 30°C.

Centrifuge for 1 min at 2,000 x g and transfer the supernatant into a fresh tube.

Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubate while shaking at 65°C overnight.

Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.

The DNA can be purified using a PCR purification kit or phenol:chloroform extraction.

DNA levels are quantitatively measured by real-time PCR.