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Detailed procedure and tips for cross-linking ChIP using ChIP-seq and ChIP-qPCR methods.
Reviewed November 9, 2021
ChIP-seq and ChIP-qPCR are powerful tools that allow the specific matching of proteins or histone modifications to regions of the genome. After the isolation of chromatin, 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. The output DNA produced using this protocol can be analyzed using either qPCR (ChIP-qPCR) or sequencing (ChIP-seq).
Formaldehyde is used to cross-link the proteins to the DNA. Cross-linking is a time-dependent procedure and optimization will be required.
Tip: We 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.
1.1 Start with two confluent 150 cm2 dishes (1x107 - 5x107 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.
1.2 Add glycine to a final concentration of 125 mM to the media and incubate with shaking for 5 min at RT.
1.3 Rinse cells twice with 10 mL cold PBS.
1.4 Add 5 mL of cold PBS, scrape dishes thoroughly with a cell scraper, and transfer into 50 mL tube.
1.5 Add 3 mL PBS to dishes, scrape again, and transfer the remainder of the cells to the 50 mL tube.
1.6 Centrifuge for 5 min, 4°C, 1,000 x g.
1.7 Carefully aspirate off supernatant and resuspend the pellet in ChIP lysis buffer (750 μL per 1x107 cells) and incubate for 10 min on ice.
Tip: When using suspension cells, start with 1x107 - 5x107 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).
2.1 Sonicate lysate to shear DNA to an average fragment size of 200 - 1000 bp. This will need optimizing as different cell lines require different sonication times.
Tip: 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.
2.2 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.
2.3 Remove 50 μL of each sonicated sample to determine DNA concentration and fragment size.
Tip: The sonicated chromatin can be snap frozen in liquid nitrogen and stored at -80°C for up to 3 months. Avoid multiple freeze-thaws.
Image 1 (above): 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.
Tip: Sonicating for too long will disrupt nucleosome-DNA interactions therefore the band size should not be smaller than 200 bp.
The sonicated chromatin samples can be used to calculate the DNA concentration for subsequent IPs and measure DNA fragment size.
3.1 Add 70 µL of elution buffer to the 50 µL of chromatin.
3.2 Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubate while shaking at 65°C overnight.
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.
3.3 Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.
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.
3.4 Purify DNA using a PCR purification kit or phenol: chloroform extraction.
3.5 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.
4.1 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 it at -20°C until further use.
4.2 Add the 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.
4.3 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.
4.4 Add 60 µL of blocked protein A/G beads to all samples and IP overnight with rotation at 4°C.
Tip: 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.
4.5 Centrifuge the immunoprecipitated samples for 1 min at 2,000 x g and remove the supernatant.
4.6 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.
Tip: If the high background is observed additional washes may be needed. Alternatively, the sonicated chromatin may 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.
5.1 Elute DNA by adding 120 μL of elution buffer to the protein A/G beads and vortex slowly for 15 min at 30°C.
5.2 Centrifuge for 1 min at 2,000 x g and transfer the supernatant into a fresh tube.
5.3 Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubate while shaking at 65°C overnight.
5.4 Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.
5.5 The DNA can be purified using a PCR purification kit or phenol-chloroform extraction.
6.1 DNA levels are often measured by real-time quantitative PCR. Primers and probes are often designed using software provided with the real-time PCR apparatus or using an online design tool. A selection of pre-designed primers and probes is available on our website.
6.2 Alternatively, analysis can be done by sequencing when using the ChIP-seq method. DNA produced using this protocol is suitable for use as an input for sequencing library prep.
Tip: We recommend using our troubleshooting tips to optimize this protocol for your particular experimental conditions.
|Species||Immunoglobin isotype||Protein A binding strength||Protein G binding strength|
|IgM||Use Anti-Human IgM||Use Anti-Human IgM|
|IgM||Use Anti-Human IgM||Use Anti-Human IgM|
|Guinea Pig||All isotypes||+++||++|