This method can help to provide key information in the field of oncology such as the identification of putative target genes and pathogenic mechanisms to develop potential therapeutic interventions. The main advantage of this technique is that one can detect the interaction of NFAT2 and other transcription factors with known and novel target genes. Generally individuals new to this method will struggle because optimal fixation and shearing conditions are difficult to establish.
To begin fill a 1.5-milliliter tube with one milliliter of 37%formaldehyde. Then fill another 1.5-milliliter tube with one milliliter of 1.25 molar glycine in a five-milliliter tube with four milliliters of PBS. Next, after the stimulation of the cells spin the cells according to the text protocol, and re-suspend the cells in 500 microliters of PBS and place the tube in an ice-filled box.
Add 13.5 microliters of 37%formaldehyde to the cells and mix by pipetting up and down. Then incubate the suspension at room temperature. After this, add 57 microliters of 1.25 molar glycine to stop the fixation.
And allow the suspension to incubate at room temperature for five minutes. Immediately following the incubation period place the cells on ice and centrifuge the cells at 500 times G for five minutes at four degrees Celsius. Then aspirate the supernatant.
Next, wash the cells twice with one milliliter of ice-cold PBS at 200 times G for five minutes at four degrees Celsius and remove the supernatant. Resuspend the cell pellet in five milliliters of lysis buffer one by pipetting up and down. Then put the samples on ice and incubate them with shaking for 10 minutes.
After this, centrifuge the tubes at 500 times G for five minutes at four degrees Celsius. Then use a pipette to carefully remove the supernatant. Homogenize the cells in five milliliters of lysis buffer two and pipette up and down to mix.
Then incubate the cells on ice for 10 minutes with shaking. After the incubation period centrifuge the tubes at 500 times G for five minutes at four degrees Celsius, and carefully remove the supernatant. Next, resuspend the cell pellet in shearing buffer one with 1X protease inhibitor.
Then incubate the cell suspension on ice for 10 minutes. Transfer 140 microliters of the cell suspension to sonicator tubes taking care to avoid forming air bubbles. Place the tubes in a focused-ultrasonicator at seven degrees Celsius and shear for 10 to seven and 1/2 minutes.
Transfer the cell suspension into 1.5-milliliter tubes. Centrifuge the samples at 15, 700 times G for 10 minutes at four degrees Celsius, and collect the supernatant in a new tube. First, prepare 20 microliters of protein A-coated beads for each precipitation in one 1.5-milliliter tube.
Allow the beads to settle on a magnetic rack for one minute, and remove the supernatant. Then wash the beads with 40 microliters of 1X chip buffer one for each 20 microliters of protein A-coated beads by pipetting up and down. Allow the beads to settle on a magnetic rack for one minute and remove the supernatant.
Repeat this washing process three more times before re-suspending the beads in their original volume of 1X chIP buffer one. Add BSA protease inhibitor, 5X chIP buffer, and ChIP-seq grade water to the beads. Next, add the sheared chromatin.
Use 10 micrograms of anti-NFAT2 antibody to capture NFAT2, and 2.5 micrograms of IgG antibody as a control. Incubate the mixtures overnight at four degrees Celsius on a wheel rotating at six RPM. The next day spin the tubes for five seconds at 7, 000 times G and incubate them on the magnetic rack for one minute.
Then aspirate the supernatant and wash the beads once with wash buffers one, two, three, and four consecutively. Spin the tubes for five seconds at 7000 times G before incubating the tubes on the magnetic rack for one minute. After this, remove the supernatant and add the next wash buffer.
After the last wash, spin the tubes for five seconds at 7000 times G.Incubate the tubes on the magnetic rack for one minute. Remove the supernatant and take up the beads in 100 microliters of elution buffer one. Then incubate the tubes on the rotating wheel for 30 minutes.
Next, briefly spin the tubes and place them on a magnetic rack for one minute. Transfer the supernatant to a new 1.5-milliliter tube and add four microliters of elution buffer two. To create the input control mix one microliter of the sheared chromatin with 99 microliters of elution buffer one and four microliters of elution buffer two.
Then incubate the samples for four hours at 65 degrees Celsius with shaking. After this, add 100 microliters of 100%isopropanol to the tubes. Vortex and spin the samples for five seconds at 7000 times G.Then add 10 microliters of magnetic beads to each sample.
Vortex and incubate the samples on a rotating wheel at room temperature for an hour. Next, spin the tubes briefly and place them on a magnetic rack for one minute. Aspirate the supernatant and resuspend the beads in 100 microliters of wash buffer one.
Invert the tubes to mix and incubate them for five minutes on a rotating wheel at room temperature. Next, briefly centrifuge the tubes. Place them in a magnetic rack for one minute, and use a pipette to remove the supernatant.
Then add 100 microliters of wash buffer two. Invert the tubes to mix and incubate them for five minutes on a rotating wheel at room temperature. After this, briefly centrifuge the tubes and place them in a magnetic rack for one minute.
Remove the wash buffer two via aspiration and resuspend the beads in 55 microliters of elution buffer. To elute the precipitated DNA incubate the samples in a rotating wheel at room temperature for 15 minutes. After this, spin the tubes briefly and place them in a magnetic rack for one minute.
Finally, transfer the supernatant to new 1.5-milliliter tubes. This protocol was first performed with Jurkat cells analyzing LCK as a potential NFAT2 target. As shown here, optimal results were documented by significant enrichment of the IL-2 positive control and LCK DNA.
Suboptimal shearing or missing fixation may lead to poor quality DNA. Finally, this protocol was performed with primary human CLL cells with CD40L serving as the positive control and LCK as the experimental target. As demonstrated by the strong enrichment of LCK DNA, LCK is a direct NFAT2 target in primary human CLL cells.
Inadequate shearing resulting in poor quality DNA can return suboptimal results. While attempting this procedure it's important to remember that the fixation and sonication steps are crucial for the success of this method and might have to be adjusted to the cells being analyzed.
