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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 efficiently bind transcription factors to DNA within your chromatin sample. This type of double cross-linking is very effective when you are 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.
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 would suggest cross-linking the samples for with EGS for 20–30 min, combined with a 10-minute formaldehyde treatment. 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.
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 g). Wash 3 times with cold PBS and resuspend pellet in ChIP Lysis Buffer (750 μL per 1x107 cells). Proceed to Step 2.
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.
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
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.
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.
If high background is observed additional washes or washes with buffers with higher salt concentrations (up to 500mM NaCl) may be needed. Alternatively, the sonicated chromatin may also be pre-cleared by incubating with the Protein A/G beads for 1 hr 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 reverse cross-links
A selection of pre-designed primers and probes are also available on our website. 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 200bp.