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

ChIP is a powerful tool that uses isolated chromatin and antibodies to the antigen of interest to determine whether a target binds to a specific DNA sequence or to map the distribution across the genome (ChIP-seq).

This protocol provides specific details of how ChIP can be performed on cells using a dual cross-linking method to bind transcription factors to DNA within your chromatin sample efficiently. This type of double cross-linking is very effective when using ChIP to observe the binding pattern of transcription factors bound directly to DNA or even those found in DNA binding complexes not bound directly to DNA.

Solutions

​ChIP buffer

• 50 mM HEPES-KOH pH7.5
• 140 mM NaCl
• 1 mM EDTA pH8
• 1% Triton X-100 or 1% NP-40
• 0.1% Sodium Deoxycholate
• 0.1% SDS
• Protease Inhibitors (add fresh each time)

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.
Cross-linking is a time-dependent procedure, and optimization will be required. We suggest cross-linking the samples with EGS for 20–30 min, combined with a 10-minute formaldehyde treatment.

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.

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

• Final % should be 0.75% for histone projects or 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.

Steps

Start with two confluent dishes

• Start with two confluent 150 mm² dishes (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.

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

• Final % should be 0.75% for histone projects or 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.

Pellet cells by centrifugation.

• We recommend centrifuging at 1,000g for 5 mins.

Wash 3 times with cold PBS and resuspend pellet in ChIP lysis buffer.

• We recommend 750 μL of lysis buffer per 1x107 cells.

Stage 2 - Sonication

Steps

Sonicate lysate to shear DNA to an average fragment size of 200–1000 bp.

• The fragment size should be analyzed on a 1.5% agarose gel, as demonstrated in Figure 1.

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

The sonicated chromatin samples can be used to calculate the DNA concentration for subsequent IPs and measure DNA fragment size.

Steps

Add 70 μL of elution buffer to the 50 μL of chromatin.

Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL).

• Incubate while shaking at 65°C overnight.

Add 2 µL proteinase K (20 mg/mL).

• Incubate while shaking at 60°C for 1 h.

Purify DNA using a PCR purification kit or phenol:chloroform extraction.

To determine the DNA concentration, transfer 5 μL of the purified DNA into a tube containing 995 μL TE.

• This should give a 200-fold dilution and you can read the OD260.
• Run purified DNA in a 1.5% agarose gel with a 100 bp DNA marker to determine fragment size.

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.