The overall goal of the following experiment is to detect either binding of DNA binding proteins such as transcription factors or his stone modifications. This is achieved by first preparing the chromatin as a second step. A chromatin immunoprecipitation reaction is set up to pull down fragments of DNA bound to the protein of interest.
Next, the DNA fragment bound to the protein of interest is amplified by PCR. Results are obtained that show fold enrichment of the DNA fragment bound to protein of interest compared to an unbound region based on QPCR analysis. This method can help answer key questions in the fields of transcription, chromatin, and epigenetics, such as when and where different modified or unmodified proteins are present.
On the DNA Though this method can provide insight into T-cell gene regulation. It can also be applied to other systems such as B lymphocytes, leukemia samples, and immortalized lines of cells. The chromatin for this experiment will be prepared from mouse splenic naive CD four T-cells.
To begin this procedure, add 37%formaldehyde to a final concentration of 1%to the CD four T-cell suspension in DMEM and gently rock at room temperature for 15 minutes. This will crosslink the DNA protein complexes. Stop the crosslinking by adding one molar glycine to a final concentration of 125 millimolar.
Continue to rock for five minutes at room temperature. Collect the cells by centrifugation at 200 G for five minutes at four degrees Celsius. Remove the supernatant and resuspend cells in five milliliters of ice cold PBS containing protease inhibitors centrifuge again, wash cells in this way for a total of three times after the final wash, discard the supernatant and resuspend cells in one milliliter of ice cold cell lysis buffer containing protease inhibitors.
Transfer the cell suspension to a 1.7 milliliter micro centrifuge tube and incubate on ice for 15 minutes. Collect the nuclei by centrifugation at 200 G for five minutes at four degrees Celsius. Carefully discard the supernatant resuspend the nuclei in 500 microliters of nuclear lysis.
Buffer containing protease inhibitors incubate on ice for 15 minutes. Next, sonicate the cells using a 1.6 millimeter probe and an output level of four sonicate for 15 seconds. Cool the sample on ice for one to two minutes to prevent overheating and sonicate again for 15 seconds.
Repeat the sonication and cooling for a total of four times centrifuge, the sonic chromatin at 16, 000 G for five minutes. At four four degrees Celsius. Collect the clear supernatant in a new micro centrifuge tube.
Remove a 20 microliter aliquot of clear supernatant. Add DNA loading dye and check the sonicated DNA by electrophoresis through a 2%agro gel. The ideal size of sonicated DNA for most applications is 200 to 500 base pair.
Finally, determine DNA concentration using a UV spectrophotometer. Sheared chromatin can be used immediately to set up a chromatin immunoprecipitation reaction or stored at negative 80 degrees Celsius for chromatin immunoprecipitation or chip dilute sonicated chromatin to A DNA concentration of five to 10 micrograms per milliliter in a total volume of two milliliters in chip dilution buffer with protease inhibitors. All the steps in this procedure must be carried out at zero to four degrees Celsius.
Save 10%which is 200 microliters in this example of the diluted sonicated chromatin as input store on ice of the remaining sample aliquot 450 microliters into each of four 1.7 milliliter microview tubes labeled as isotype control and antibody of interest. If multiple antibodies of the same isotype are used, a single isotype control will be sufficient To perform this analysis, add two to five micrograms of specific antibody depending on the specificity of the antibody used or isotype control to the respective tubes. Rock the tubes overnight at four degrees Celsius to allow formation of chromatin antibody complexes on the following morning.
Add 25 microliters of protein G magnetic beads to each mixture and rock for at least two hours at four degrees Celsius. Next place the fuge tubes on a magnetic stand for 15 to 20 seconds to allow the beads to collect on the magnetized side. Carefully remove the solution by aspiration without disturbing the beads.
Add one milliliter of low salt wash solution and gently rock for five minutes on the mutator. Collect the beads using the magnetic stand and remove the wash solution. Add one milliliter of low salt wash solution again and gently rock for five minutes.
After removing the low salt wash solution, add one milliliter of high salt wash solution and rock for five minutes. Collect the beads using the magnetic stand and remove the wash solution. Repeat the wash with the high salt wash solution.
Once remove the high salt wash solution, add one milliliter of lithium chloride wash solution, and rock for five minutes. Collect the beads using the magnetic stand and remove the wash solution. Repeat the lithium chloride wash once.
Add one milliliter of TE solution and rock for five minutes. Collect the beads using the magnetic stand and remove the wash solution. Elute the DNA from the beads by adding 250 microliters of elucian buffer rock for 15 minutes at room temperature.
Pipet off the EIT and save this material in a new 1.7 millimeter fuge tube. Repeat once more and combine both the elu, discard the beads to reverse the cross links. Add to the eluded DNA five molar sodium chloride to a final concentration of 0.3 molar and one microliter of RNAs.
A add 400 microliters of elution buffer to the inputs saved earlier. To make the volume 500 microliters to each input, add sodium chloride to 0.3 molar one microliter of RNAs, a 10 microliters of 0.5 molar EDTA 20 microliters of one molar tris, HCL pH 6.5 and one microliter of proteinase K.Seal the tubes and incubate them overnight at 65 degrees Celsius in a dry heating block on the following morning, let the tubes cool to room temperature. Then add one milliliter of 100%ethanol and incubate for two hours to overnight at negative 80 degrees Celsius.
To precipitate the DNA centrifuge, the tubes at 16, 000 G for 15 minutes. To pellet the DNA wash, the DNA pellet once with 70%ethanol and air dry, the pellet resuspend, the DNA pellet in 100 microliters of autoclave distilled water, purify DNA using kayak, quick spin columns and elute in a total volume of 50 microliters elution buffer. This DNA is now ready for use for PCR amplification.
The analysis of the QPCR reserves to obtain full enrichment over a control unbound region is the most important aspect of this protocol. Using the QPCR software, go to the editor and mark the wells containing input samples of various dilutions with their standard curve values. Also label the wells with unknown chip samples exactly as the PCR plate is laid out.
The use of a standard curve to interpolate DNA amounts in the unknown specific and isotype samples allows the evaluation and matching of the quality and efficiency of all primer sets over the range of concentrations found in the samples. Go to the subset editor and mark the subsets, including the wells with input samples as well as the unknown chip samples. The unknown samples will be quantified using the standard curve generated from the known concentrations of input samples for the analysis.
After the PCR amplification is complete, perform the analysis with ABS quant. Second derivative max. Select the subset for analysis and press.Okay.
Press calculate, which will generate the standard curve using the known concentrations of input samples and will display the absolute quantity of DNA present in the unknown samples if the quality of she DNA is good and if the primers bind specifically to the targeted region, PCR efficiency should be close to two. Perform a melting curve analysis with TM calling for the same subset if available. A single peak indicates that only one specific product was amplified.
Amplification of specific DNA can also be tested by electro the PCR product, along with the DNA ladder through an AROS gel. The data obtained is then exported as a tab delimited text file, which can be opened in Microsoft Excel for further analysis. Divide the DNA amount for the specific antibody with the isotype control for targeted primers.
Repeat this step for the control region Primers. If multiple antibodies of the same isotype are used for different immuno precipitations normalize each of these with the values from the same isotype control. Moreover, if multiple targeted primer pairs are used, the DNA quantities from a single control primer repair set should be used for normalization of each target.
The output values represent the specific antibody immunoprecipitation fold enrichment at each location relative to nonspecific antibody Background immunoprecipitation divide the fold enrichment for targeted primers with the fold enrichment for control region primers to obtain enrichment relative to a control unbound region. Importantly, note that because of the generation of standard curves with total input DNA for each sample, each of the immunoprecipitation values interpolated from the standard are already normalized to and expressed as the fraction of total input by the machine software. The chromatin immunoprecipitation protocol demonstrated here controls for differences, if any, in the amount of DNA used in PCR through use of a primer pair that amplifies an unbound region of genome, thus serving as a loading control.
In this example, the coding region of the mouse beta actin gene was used as a region unbound to the protein of interest and the transcription factor and fat binding site on the mouse, IL two promoter was used as a target region. This snapshot of an Excel spreadsheet depicts the use of the calculation protocol described in the analysis data from three experimental repeats in the boxes colored yellow, green, and purple with three technical replicates. Each was used to calculate fold enrichment.
Relative enrichment of a protein bound to different regions of the genome can be compared if the same set of isotype control and specific antibodies are chosen. If the specificity of the antibody is good, a two to tenfold enrichment over the unbound control region is typically observed. This figure shows the results of a chip experiment with conventional CD four T cells and regulatory CD four T cells isolated from mice.
A small fraction of conventional T cells was stimulated with PMA and ion mycin for 30 minutes. Relative enrichment of NFA transcription factors, N fat C one, and NFA C two at the IL two promoter was observed and is depicted here as fold enrichment over an unbound region Following this procedure. Other methods such as ChIP-seq can be used in order to address global occupancy of transcription factors or changes globally in epigenetic marks in response to a stimulus.
After watching this video, you should have a good understanding of how to perform chromatin immunoprecipitation using a limited number of cells, and to analyze QPCI data to determine transcription factor banding compared to a control unbound region.
