All tags Epigenetics Cross-linking chromatin immunoprecipitation (X-ChIP) protocol

Cross-linking chromatin immunoprecipitation (X-ChIP) protocol

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Detailed procedure and tips for cross-linking ChIP.  

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View the X-ChIP protocol diagram.

ChIP is a powerful tool that allows the specific matching of proteins or histone modifications to regions of the genome. Chromatin is isolated and antibodies to the antigen of interest are used to determine whether the target binds to a specific DNA sequence or to map the distribution across the genome (microarray or DNA sequencing). This can be performed both spatially and temporally. This protocol provides specific details of how ChIP can be performed on cells. 

  1. Cross-linking and cell harvesting



    Formaldehyde is used to cross-link the proteins to the DNA. Cross-linking is a time dependent procedure and optimization will be required. We would suggest cross-linking the samples for 2–30 min. Excessive cross-linking reduces antigen accessibility and sonication efficiency. Epitopes may also be masked. Glycine is added to quench the formaldehyde and terminates the cross-linking reaction.



    1. Start with two confluent 150 cm2 dishes (1x1075x107 cells per dish). Cross-link proteins to DNA by adding formaldehyde drop-wise directly to the media to a final concentration of 0.75% and rotate gently at room temperature (RT) for 10 min.
    2. Add glycine to a final concentration of 125 mM to the media and incubate with shaking for 5 min at RT.
    3. Rinse cells twice with 10 mL cold PBS.
    4. Add 5 mL of cold PBS, scrape dishes thoroughly with a cell scraper and transfer into 50 mL tube.
    5. Add 3 mL PBS to dishes, scrape again and transfer the remainder of the cells to the 50 mL tube.
    6. Centrifuge for 5 min, 4°C, 1,000 x g.
    7. Carefully aspirate off supernatant and resuspend the pellet in ChIP lysis buffer (750 μL per 1x107 cells) and incubate for 10 min on ice.



      When using suspension cells, start with 1x1075x107 cells and treat with both 0.75% formaldehyde and glycine as described above (Step 1). Pellet cells by centrifugation (5 mins, 1,000 x g). Wash 3 times with cold PBS and resuspend pellet in ChIP lysis buffer (750 μL per 1x107 cells). Proceed to Step 2.


  2. Sonication

    1. Sonicate lysate to shear DNA to an average fragment size of 2001000 bp. This will need optimizing as different cell lines require different sonication times.



      The cross-linked lysate should be sonicated over a time-course to identify optimal conditions. Samples should be removed over the time-course and DNA isolated as described in Step 3. The fragment size should be analyzed on a 1.5% agarose gel as demonstrated in Figure 1.


    2. After sonication, pellet cell debris by centrifugation for 10 min, 4°C, 8,000 x g. Transfer supernatant to a new tube. This chromatin preparation will be used for the immunoprecipitation (IP) in Step 4.
    3. Remove 50 μL of each sonicated sample, to determine DNA concentration and fragment size.



      The sonicated chromatin can be snap frozen in liquid nitrogen and stored at -80°C for up to 3 months. Avoid multiple freeze-thaws.


  3. Determination of DNA concentration and fragment size

    1. The sonicated chromatin samples can be used to calculate the DNA concentration for subsequent IPs and measure DNA fragment size. Add 70 µL of elution buffer to the 50 µL of chromatin.
    2. Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL)  and incubate while shaking at 65°C overnight.
    3. Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h​. 



      Samples are treated with RNase A as high levels of RNA will interfere with DNA purification when using the PCR purification kit. Yields can be severely reduced as the columns become saturated.

      Samples are treated with proteinase K, which 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.
    4. Purify DNA using a PCR purification kit or phenol:chloroform extraction.
    5. To determine the DNA concentration, transfer 5 μL of the purified DNA into a tube containing 995 μL TE to give a 200-fold dilution and read the OD260. The concentration of DNA in μg/mL is OD260  x 10,000. This is used to calculate the DNA concentration of the chromatin preparation. Run purified DNA in a 1.5% agarose gel with a 100 bp DNA marker to determine fragment size.
  4. Immunoprecipitation

    1. Use the chromatin prepared from Step 2.2. Approximately 25 μg of DNA per IP is recommended. 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 at -20°C until further use.
    2. Add primary antibody to all samples except the beads-only control and rotate at 4°C for 1 h. The amount of antibody to be added should be determined empirically; 1–10 μg of antibody per 25 μg of DNA often works well.
    3. 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 single stranded 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.
    4. Add 60 µL of blocked protein A/G beads to all samples and IP overnight with rotation at 4°C.



      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.


    5. Centrifuge the immunoprecipitated samples for 1 min at 2,000 x g and remove the supernatant.
    6. Perform the following washes: once in low salt wash buffer, once in high salt wash buffer, once in LiCl wash buffer. After each wash, centrifuge for 1 min at 2,000 x g and remove the supernatant.



      If high background is observed additional washes may be needed. Alternatively, the sonicated chromatin may also be pre-cleared by incubating with the Protein A/G beads for 1 h prior to Step 4.2. Any non-specific binding to the beads will be removed during this additional step. Transfer the supernatant (sonicated chromatin) to a new tube and incubate with the antibody and beads as described in Step 4.2 onwards.


  5. Elution and reversal of cross-links

    1. Elute DNA by adding 120 μL of elution buffer to the protein A/G beads and vortex slowly for 15 min at 30°C.
    2. Centrifuge for 1 min at 2,000 x g and transfer the supernatant into a fresh tube.
    3. Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL)  and incubate while shaking at 65°C overnight.
    4. Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.
    5. The DNA can be purified using a PCR purification kit or phenol:chloroform extraction.
    6. DNA levels are quantitatively measured by real-time PCR. Primers and probes are often designed using software provided with the real-time PCR apparatus. Alternatively an online design tool is used.

A selection of pre-designed primers and probes are also available on our website.

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Please use our troubleshooting tips to optimize the protocol.


Figure 1. U2OS cells were sonicated for 5, 10, 15 and 20 min. The fragment size decreases during the time course. The optimal fragment size is observed at 15 min. NOTE; sonicating for too long will disrupt nucleosome-DNA interactions therefore the band size should not be smaller than 200 bp.

Immunoglobulin isotypes

Species Immunoglobulin isotypeProtein AProtein G
Human          IgG1++++++
                       IgG2++++++
                       IgG3-+++
                      IgG4++++++
                      IgMUse anti Human IgMUse anti Human IgM
                       IgE-+
                       IgA-+
Mouse         IgG1++++
                      IgG2a++++++
                      IgG2b++++
                       IgG3++
                       IgMUse anti human IgMUse anti human IgM
Rat                 IgG1-+
                       IgG2a-+++
                       IgG2b-++
                       IgG2c+++
Chicken        all isotypes-++
Cow              all isotypes+++++
Goat              all isotypes-++
Guinea Pig  all isotypes+++++
Hamster     all isotypes+++
Horse         all isotypes+++++
Pig              all isotypes+++
Rabbit        all isotypes+++++
Sheep        all isotypes-++

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

Solutions

ChIP Lysis Buffer
50 mM HEPES-KOH pH7.5  
140 mM NaCl    
1 mM EDTA pH8   
1% Triton X-100   
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)

Low Salt Wash Buffer
0.1% SDS    
1% Triton X-100   
2 mM EDTA  
20 mM Tris-HCl pH 8.0
150 mM NaCl    

High Salt Wash Buffer
0.1% SDS    
1% Triton X-100   
2 mM EDTA   
20 mM Tris-HCl pH 8.0 
500 mM NaCl  

LiCl Wash Buffer
0.25 M LiCl
1% NP-40
1% Sodium Deoxycholate
1 mM EDTA
10 mM Tris-HCl pH 8.0

TE Buffer
10 mM Tris pH 8.0
1 mM EDTA

Elution Buffer
1% SDS    
100mM NaHCO3  


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