Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

In this protocol, we can identify the interactions between proteins and DNA, revealing the transcriptional regulation of the gene. This helps to understand the molecular mechanisms in the cells. We modify the pre-existing protocols and then establish a straightforward and clearer assay protocols.

You just simply follow the protocol from the first step so that the assay can be done successfully. To prepare crosslinked chromatin, in a fume hood, add four milliliters of 1%VCaP cell culture media and 0.229 milliliters of 18.5%PFA to a six-centimeter dish. Gently swirl the dish to distribute the PFA evenly.

Incubate at room temperature for exactly 10 minutes. Then, add 0.47 milliliters of 1.25-molar glycine solution to the dish, and incubate at room temperature for five minutes to stop further crosslinking. After washing the cells according to the manuscript, scrape the cells and transfer the cell suspension to a 1.5-milliliter microcentrifuge tube.

Centrifuge the tube with the cell suspension at 3, 000 times g for five minutes at room temperature to recover the cell pellet. Use a pipette to completely remove the PBS. Record the cell number per tube, and store the cell pellet at minus 80 degrees Celsius.

To perform cell lysis, first thaw the stored VCaP cell pellets on ice. Add 300 microliters of the prepared ChIP cell lysis buffer supplemented with PIC to each tube, and resuspend the pellet thoroughly. Vortex the tube for 15 seconds, and incubate the suspension on ice for 10 minutes.

Centrifuge at 9, 000 times g for three minutes at four degrees Celsius. Remove the supernatant completely, and add 300 microliters of of MNase digestion buffer to resuspend the pellet. Dilute MNase with MNase digestion buffer in a 1.5-milliliter tube to yield 50 gel units per microliter.

Add the diluted MNase to the suspension, and incubate at 37 degrees Celsius for exactly 10 minutes. Set the mixing by inversion every 2 1/2 minutes. To terminate the MNase digestion, add 30 microliters of 0.5-molar EDTA at pH eight, and vortex briefly.

After incubation for five minutes on ice, centrifuge at 9, 000 times g for five minutes at four degrees Celsius. Remove the supernatant completely, and resuspend the pellet in 300 microliters of the prepared ChIP dilution buffer supplemented with PIC. Then, on a sonicator equipped with a microtip probe, set the sonication conditions to amplitude two, processed time 15 seconds, pulse ON five seconds, and pulse OFF 30 seconds.

Sonicate the suspension on ice. Draw one microliter of the suspension, and spot onto a slide glass. Under a microscope, observe to ensure that the cell structure is almost broken.

Centrifuge the tube at 9, 000 times g for 10 minutes at four degrees Celsius, and transfer the supernatant to a new 1.5-milliliter microcentrifuge tube. Save 20 microliters of the digested chromatin in a 1.5-milliliter screw tube for further treatment, and store the remainder at minus 80 degrees Celsius. First, in each 1.5-milliliter screw tube, add 75 microliters of water, four microliters of five-molar sodium chloride, and one microliter of proteinase K.To remove crosslinking between protein and DNA, add 20 microliters of digested chromatin from each MNase digestion condition to a screw tube.

Close the cap tightly, mix completely, and incubate the tube at 65 degrees Celsius overnight. In the morning, after centrifuging the tube at two to 3, 000 times g for a few seconds, add 100 microliters of PCI. Vortex vigorously to form an emulsion.

Centrifuge the tube again at maximum speed for 30 seconds at room temperature. Then, prepare two 1.5-milliliter microcentrifuge tubes per condition. Add 100 microliters of PCI to one tube.

Add 10 microliters of three-molar sodium acetate at pH 5.2 and two microliters of glycogen to another tube. From the tube taken out of the centrifuge, carefully draw the upper phase containing DNA, and add it to the tube containing PCI. Vortex vigorously, and centrifuge the tube at maximum speed for 30 seconds at room temperature.

Next, transfer the upper phase to the tube containing sodium acetate and glycogen prepared previously. Add 250 microliters of ethanol, and mix by inversion. Incubate for 10 minutes at room temperature.

Centrifuge the tube at maximum speed for 30 minutes at four degrees Celsius. Confirm the pellets on the bottom of the tube. Carefully remove the supernatant so as not to disturb the pellet, and add 500 microliters of 70%ethanol.

Centrifuge the tube at maximum speed for five minutes at four degrees Celsius. Remove the supernatant completely, and dry the pellet for approximately five minutes at room temperature. Then, add 20 microliters of TE to dissolve the pellet.

Measure the DNA concentration using a UV spectrophotometer. After preparation of digested chromatin, thaw all samples on ice. Add 1200 microliters of the prepared ChIP dilution buffer supplemented with PIC to dilute digested chromatin to the concentration of five micrograms per 500 microliters.

Add five microliters of the diluted digested chromatin to a 1.5-milliliter screw tube as an input sample. Store the sample at minus 80 degrees Celsius. Add 500 microliters each of the diluted digested chromatin to a 1.5-milliliter screw tube as an immunoprecipitation sample.

Add two micrograms of antibody to each tube, and close the cap. Incubate the tubes at four degrees Celsius overnight with gently mixing on a rocking platform. In the morning, add 30 microliters of ChIP-grade protein G magnetic beads to each tube.

Incubate the tubes again at four degrees Celsius for two hours with gentle mixing. After that, spin down the tube briefly at two to 3, 000 times g, and place the tube in a polyethylene rack containing neodymium magnets for one minute. Then, carefully remove the supernatant by aspiration.

Next, add 0.5 milliliters of low-salt immune complex wash buffer to each tube. Briefly vortex the tube to disperse the beads. Incubate the tube at four degrees Celsius for five minutes with gentle mixing on a rocking platform.

Repeat the wash with high-salt immune complex and lithium chloride immune complex according to the manuscript. After removing the remaining supernatant completely, add 150 microliters of elution buffer to the tube. Vortex the tube to disperse the beads completely.

Close the cap, and incubate the tube at 65 degrees Celsius for 30 minutes. To disperse the beads thoroughly, mix by inversion every five minutes. During the incubation, prepare a 1.5-milliliter screw tube, and add six microliters of five-molar sodium chloride and two microliters of proteinase K.Thaw the 1%input sample previously prepared on ice.

Add 150 microliters of elution buffer, six microliters of five-molar sodium chloride, and two microliters of proteinase K to the input sample. After the incubation of the tube containing beads, spin down. Place the tube in a magnetic rack for one minute, and transfer the supernatant to the screw tube containing sodium chloride and proteinase K.Representative microphotographs of crosslinked cell pellets before and after sonication show distinct differences.

Without sonication, the cell structure remains intact, indicating that the chromatin is present in the nuclei. A brief sonication breaks the cell structure. Crosslinked chromatin was prepared from VCaP cells and digested with various amounts of MNase.

Without adding MNase, a smear pattern with a very high molecular weight appeared. The addition of MNase gave a ladder pattern, which shows that MNase digests internucleosome. Only the condition that produced chromatin fragments up to 900 bp can be considered proper digestion, whereas an inappropriate digestion pattern shows overdigestion mainly resulted in mononucleosome production.

Digestion pattern of chromatin in VCaP cells indicate proper digestion of chromatin. The highest occupancy of H3K4methyl3 was observed around 0.5 kilobase and one kilobase upstream of the AR-TSS. Regions located at 19 kilobase and eight kb upstream and 12 kilobase downstream of AR-TSS, however, had little occupancy of H3K4methyl3, indicating that these can be used as negative regions.

Determining MNase digestion condition is most important. Maintain cell number, buffer volume, and enzyme amount in every experiment. The condition must be identified for each cell type.

We can perform ChIP sequencing for global occupancy of the factors and chromatin conformation captures to determine the interactions between distal genomic lesions. This method could reveal process mechanisms of the transcription. ChIP assay is a powerful tool for investigating transcriptional regulation, but it is cumbersome and not reproducible.

Our method encourage researchers to carry out the assay routinely and easily.