The overall goal of this protocol is to efficiently perform chromatin immunoprecipitation in mouse T-cell lines with high reproducability in a reasonable time to study the distribution of histone marks at specific genomic regions. This method can help answer key questions in the chromatin biology field with particular focus on T-cells such as how chromatin structure is regulated during T-cell dividement or activation. The main advantage of this technique is that the chromatin shearing is efficient and highly reproducible.
Visual demonstration of this method is critical as the chromatin shearing conditions are difficult to establish as the phenol-chloroform isoamylic alcohol steps can be challenging. Demonstrating the procedure will be Francesca Ferrante, a PhD student in our laboratory. On day one, transfer the mouse T-cells from a six well plate to a 50 milliliter tube.
Then centrifuge the cells for five minutes at 24 degrees celsius at approximately 271 times G.After resuspending the cell pellet in 30 milliliters of IMDM cell culture medium, count the cells using a Neubauer chamber. Collect aliquots of 20 million cells into new 50 milliliter tubes. Then add formaldehyde to a 1%final concentration directly to the cell culture medium and incubate at room temperature for ten minutes.
Next add a 1/8 volume of one molar glycine pH 7.5 and incubate at room temperature for five minutes. Pellet the cells by centrifugation for five minutes at 24 degrees celsius and approximately 271 times G.Then wash the cells by resuspending the cell pellet in 10 milliliters of phosphate buffered saline and repeating the centrifugation. Wash the cells again with one milliliter of PBS.
Transferring the resuspended cells to 1.5 milliliter tubes before centrifuging. The shearing of the chromatin is the most critical step as depends on the amount of cells, shearing tubes, device, and lysis buffer as well as shearing parameters. Following the wash, resuspend the cell pellet in one milliliter of SGS lysis buffer and incubate on ice.
After 10 minutes, transfer each sample to a sonication tube. Then perform shearing using a focused-ultrasonicator according to the settings indicated in the text protocol. After shearing, transfer the samples to 1.5 milliliter tubes.
Centrifuge for 10 minutes at four degrees celsius and approximately 18, 000 times G.Then transfer the supernatant to new tubes. Collect 50 microliters for determination of the shearing efficiency and snap freeze the sheared lysate in liquid nitrogen. Store the lysate in single use aliquots at minus 80 degrees celsius.
To determine the shearing efficiency, first add 50 microliters of elution buffer to the 50 microliters of each sheared lysate. Incubate the mixtures at 65 degrees celsius overnight with shaking. On day two, add 100 microliters of TE buffer, and four microliters of 10 milligrams per milliliter Rnase A to the samples.
Mix the samples and incubate for two hours at 37 degrees celsius with shaking. Then add two microliters of 20 milligrams per milliliter proteinase K to each sample, before incubating for two hours at 55 degrees celsius with shaking. Next transfer each sample to a gel tube suitable for phenol chloroform isoamyl alcohol extraction.
Handle the tubes according to the manufacturer's instructions. Add 200 microliters of phenol chloroform isoamyl alcohol and shake the tubes. Following centrifugation, transfer the upper phase to new tubes.
Repeat this process once. Purify the DNA by using purification columns according to the manufacturer's instructions, except to wash the membrane twice. After the last wash, leave the tube open for two minutes at room temperature to evaporate the residual ethanol.
For the elution, add 50 microliters of water to the column and incubate at room temperature for one minute. Then spin the tube for one second to properly wet the membrane. Incubate the membrane at room temperature for one minute before eluting the DNA by centrifugation.
Quantify the purified DNA on a flourometer using a high sensitivity kit according to the manufacturer's instructions. Finally analyze approximately 500 nanograms of the purified DNA on a 1.8%agarose gel. And approximately one nanogram of the purified DNA on an electrophoresis system using a high sensitivity kit.
On day three, dilute one volume of sheared lysate with five volumes of dilution buffer. Pre clear the diluted lysate with 30 microliters per milliliter of protein A agarose beads for 30 minutes while rotating in the cold room. Centrifuge the samples at four degrees celsius for five minutes at approximately 750 times G.Collect 10%of the cleared lysate as the input samples and store it at four degrees celsius.
Next aliquot the desired amount of the cleared lysate into new tubes. Then add the chosen antibodies to each tube and rotate overnight at four degrees celsius. On day four, add 40 microliters of protein A beads.
Incubate the mixture for one hour at four degrees celsius while rotating. Wash the beads with one milliliter of low salt buffer by incubating for five minutes at four degrees celsius while rotating. Then centrifuge for three minutes at four degrees celsius and approximately 950 times G.Repeat the wash with high salt buffer, lithium chloride buffer, and TE buffer.
Then add 110 microliters of elution buffer to the samples. Incubate them for 15 minutes at room temperature, vortexing the samples every two minutes. After centrifuging again, transfer 100 microliters of supernatant to a new 1.5 milliliter tube.
Repeat the elution with 100 microliters of elution buffer and combine the eluates. In the meantime, add elution buffer to the previously collected input samples to achieve a final volume of 200 microliters. Incubate all samples overnight at 65 degrees celsius with shaking.
On day five, add 200 microliters of TE buffer to each eluate and eight microliters of 10 milligrams per milliliter Rnase A.Mix the samples before incubating for two hours at 37 degrees celsius with shaking. Then add four microliters of 20 milligrams per milliliter proteinase K to each eluate before incubating for two hours at 55 degrees celsius with shaking. Next transfer each sample to a gel tube suitable for phenol chloroform isoamyl alcohol extraction.
Add 400 microliters of phenol chloroform isoamyl alcohol and shake the tubes. After centrifuging the tubes, transfer the upper phase to new tubes and repeat this process once. Finally, purify the DNA using purification columns as before.
This representative result of the shearing of mouse T-cell lines shows efficient fragmentation of the chromatin. Shown here is a representative chromatin immunoprecipitation experiment in mouse T-cell lines. The promoter of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase is highly enriched for trimethylation of lysine four on histone three.
And acetylation of lysine 27 on histone three. But not for monomethylation of lysine four on histone three. Conversely, the enhancer of the inactive gene deltex-1 is highly enriched for monomethylation of lysine four on histone three.
But poorly enriched for trimethylation of lysine four on histone three. And acetylation of lysine 27 on histone three. While attempting this procedure, it's important to remember to resolve SGS precipitates before shearing the samples.
We first had the idea for this method when studying gene regulation T-cells. We selected T-cells because they are highly relevant to medical applications and represent one of the best described model systems to study gene expression higher eukaryotes. After it's development, this technique paved the way for researchers in the field of chromatin biology to explore chromatin regulation in mammalian systems.
The implications of this technique extend towards therapy of T-cell associated diseases because it allows for dissection of the establishment and regulation of the T-cell gene expression programs. Don't forget that working with formaldehyde, phenol chloroform, and isoamylic alcohol is extremely serious and precautions such as working under a fume hood and wearing the proper protective equipment should always be taken while performing this procedure. Although this method can provide insights into mouse T-cell chromatin regulation, it can also be applied to other systems such as endothelial and lung cell lines.
