Dual Cross-linking ChIP Protocol

Licia Miller   Product Manager

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

Dual-crosslinking chromatin immunoprecipitation (Dual Cross-linking ChIP) is a technique used to study protein-DNA interactions by using two crosslinking agents (such as formaldehyde and EGS or DSG) to fix the interactions between proteins and DNA, thereby improving the stability and resolution of the experiment.

This protocol uses formaldehyde and EGS (ethylene glycol bis(succinimidyl succinate)) to double crosslink proteins to DNA in order to effectively bind transcription factors to DNA in chromatin samples. This double crosslinking approach is very effective for observing the binding mode of transcription factors that are directly bound to DNA or in complexes that are not directly bound to DNA.

Required reagent formula

(1) ChIP buffer

50 mM HEPES-KOH pH 7.5

140 mM NaCl

1 mM EDTA pH 8

1% Triton X-100

0.1% Sodium deoxycholate

0.1% SDS

Protease inhibitors (add fresh each time)

(2) RIPA buffer

50 mM Tris-HCl pH 8

150 mM NaCl

2 mM EDTA pH 8

1% NP-40

0.5% Sodium deoxycholate

0.1% SDS

Protease inhibitors (add fresh each time)

(3) Washing buffer

2 mM EDTA

20 mM Tris-HCl pH 8.0

150 mM NaCl

0.1% SDS

1% Triton X-100

(4) TE buffer

10 mM Tris pH 8.0

1 mM EDTA

(5) Elution buffer

1% SDS

100 mM NaHCO3

Phase 1   Cross-linking treatment

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Cross-linking is a time-dependent process that requires optimization. We recommend cross-linking samples with EGS for 20-30 minutes, combined with a 10-minute formaldehyde treatment.

Experimental Steps

1. First, prepare two 150 cm2 culture dishes filled with cells, each with 1 × 107 - 5 × 107 cells. Add 20 mL of ice-cold PBS to each dish and add 300 mM EGS to a final concentration of 1.5 mM.

2. Gently rotate at room temperature for 30 minutes to promote protein-DNA crosslinking.

Note: The cross-linking time should not be too long. Excessive cross-linking may also mask the epitope, reducing the accessibility of the antigen and the efficiency of sonication.

3. In a fume hood, add 37% formaldehyde to each flask to a final dilution of 0.75% if studying histones or 1% if studying transcription factors.

4. Rotate gently at room temperature for 10 minutes (this time needs to be optimized for different cell types).

Notice:

The time required will vary for different cell types and needs further optimization.

Because formaldehyde is toxic, the operation needs to be carried out in a fume hood.

5. In a fume hood, add 1.5 mL of 2.5 M glycine (final concentration 125 mM) to the culture medium and incubate with shaking for 5 minutes to quench the formaldehyde.

6. Rinse cells twice with 10 mL cold PBS.

7. Add 5 mL of cold PBS, scrape the dish thoroughly with a cell scraper, and transfer the cells to a 50 mL centrifuge tube.

8. Add 3 mL of PBS back into the dish, scrape again, and transfer the remaining cells to a 50 mL centrifuge tube.

9. Centrifuge at 1,000 × g for 5 minutes at 4°C.

10. Carefully aspirate the supernatant. Resuspend the cell pellet in ChIP lysis buffer, adding approximately 750 μL per 1 × 107 cells. Incubate in ChIP lysis buffer on ice for 10 min.

Phase 2   Ultrasonic treatment

Experimental Steps

1. Sonicate the lysate to shear the DNA into fragments with an average size of 200 - 1000 bp.

Note: The ultrasonic time varies for different cell lines and needs to be optimized according to the actual situation. During the ultrasonic treatment, samples can be taken out at intervals to measure the fragment size to determine the optimal treatment time.

2. After ultrasonic treatment, centrifuge at 8,000 × g for 10 min to pellet cell debris. Transfer the supernatant to a fresh tube.

Take out 50 μL from each sonicated sample and determine the DNA concentration and fragment size.

Phase 3   Determine DNA concentration and fragment size

Experimental Steps

1. Add 70 μL of elution buffer to 50 μL of chromatin. Add 4.8 μL of 5 M NaCl and 2 μL of 10 mg/mL RNase A. Incubate overnight at 65°C with shaking.

2. Add 2 µL of 20 mg/mL proteinase K and incubate at 60°C with shaking for 1 hour. This is used to break the cross-links between proteins and DNA, which helps in DNA purification.

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

4. Take 5 µL of purified DNA and add it to the EP tube containing 995 μL TE to read OD260 and determine the DNA concentration.

DNA concentration (µg /mL) equals OD260 value × 10,000.

The size of DNA fragments can be determined by 1.5% agarose gel.

Stage 4​   Immunoprecipitation

Experimental Steps

1. Dilute the prepared chromatin into RIPA buffer at a ratio of 1:10. Reserve samples for the experimental and control groups. It is recommended to use about 25 μg DNA per IP.

Take out 50 μL of chromatin as Input and store it at -20℃ before use.

2. Except for the control group containing only magnetic beads, add appropriate amount of primary antibody to all other sample groups and rotate at 4°C for 1 hour.

Typically, adding 1-10 μg of antibody per 25 μg of DNA works well.

Magnetic beads should use protein A magnetic beads , protein G magnetic beads, or a mixture of the two.

3. Preparation of Protein A/G Magnetic Beads.

3.1 If using both Protein A and Protein G magnetic beads, mix equal volumes of Protein A and Protein G magnetic beads and wash three times in RIPA buffer.

3.2 Aspirate the RIPA buffer and add herring sperm DNA to a final concentration of 75 ng/μL on the magnetic beads, and add BSA to a final concentration of 0.1 μg/μL.

3.3 Add RIPA buffer to double the volume of magnetic beads and incubate with rotation at room temperature for 30 minutes.

3.4 Wash once with RIPA buffer and add RIPA buffer to twice the volume of magnetic beads.

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

5. Centrifuge the immunoprecipitated sample at 2,000 × g for 1 minute and remove the supernatant.

6. Wash with wash buffer, centrifuge at 2000 × g for 1 minute each time, and remove the supernatant. Repeat twice.

Phase 5   Elution and reversal of crosslinks

Experimental Steps

1. Add 120 μL of elution buffer to the protein A/G magnetic beads and vortex slowly at 30°C for 15 minutes to elute the DNA.

2. Centrifuge at 2,000 × g for 1 minute and transfer the supernatant to a new tube.

3. Add 4.8 µL 5 M NaCl and 2 µL 10 mg/mL RNase A, incubate at 65°C with shaking overnight.

4. Add 2 µL of 20 mg/mL proteinase K and incubate at 60°C with shaking for 1 hour.

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

6. Quantification of DNA levels by real-time PCR.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/