The chip experiment is used to monitor dynamic changes in histo modifications that occur during STAT one activated gene expression alterations in the occupancy of transcription factors, histone modifying complexes and the transcription machinery itself can also be assayed in this way. To begin the assay cells are induced with interferon gamma to activate STAT one signaling cross-linking the cells with formaldehyde and collecting them follows proceed to isolate nucle and sonicate, which shears the chromatin into approximately 200 to 1000 base pair fragments. Next, add an antibody that targets the protein or histo modification of interest and then precipitate the immuno complexes.
The immuno complexes are washed, the cross links are reversed and the DNA is purified. The changes in histo modifications or recruitment of proteins to specific areas of a gene can be determined based on quantitative realtime PCR analysis of the immuno precipitated DNA. Hi, my name is Lauren Borow from the laboratory of Dr.Melissa Hendrickson and the Department of Biology at the University of Virginia.
Today we're gonna show you a procedure for chromatin immunoprecipitation. We use this procedure in our lab to study dynamic histo modifications that occur during transcription downstream of STAT one signaling. So let's get started.
One day before beginning this procedure, culture the cell so that a sufficient number is available for the two FTGH cells used Here. Prepare one 15 centimeter tissue culture dish at 80%co fluency for each chip assay. In each experiment include a negative IgG control, a positive pan histone antibody control and duplicates.
To begin this procedure, remove the culture media. Replace it with 20 milliliters of DMEM supplemented with 10%cosmic calf serum and containing interferon gamma at five nanograms per milliliter. Also, replace the media on the un induced plates with 20 milliliters of 10%C-C-S-D-M-E-M.
Next work in a fume hood. Tilt the 15 centimeter tissue culture dish and add 540 microliters of a 37%formaldehyde solution to 20 milliliters of media. Then rock the plate to spread it evenly.
Allow cross-linking to perceive the 10 minutes at room temperature. Then add glycine to 125 millimolar Final concentration to stop the cross-linking reaction. Vacuum aspirate the media and wash the cells once with 10 milliliters of ice cold PBS using a cell scrapper, collect the cells in around seven milliliters of PBS and combine cells that were treated identically from six 15 centimeter plates in a 50 milliliter conical tube on ice.
Finally centrifuge the collected cells at 1, 800 RPM for five minutes at four degrees Celsius and aspirate the PBS. If stopping here, snap, freeze the cell palette and store at minus 80 degrees Celsius after cross-linking. The next step is preparing the chromatin.
Start by adding one millimolar DTT and complete protease inhibitors to swelling buffer chilled on ice. Resus suspend the pellet from six plates, pulled in four milliliters of the swelling buffer and transfer to a 15 milliliter conical tube. Using more or less buffer might change the dancing efficiency.
Incubate the cell suspension on ice for 10 minutes. When the incubation on ice is complete, transfer the suspension to a pre chilled 15 milliliter downer and dance. 20 strokes with PEs A after dancing transfer the cells back to a 15 milliliter conical tube on ice centrifuge at 2, 500 RPM for five minutes at four degrees Celsius and discard the supernatant leaving the nuclear pellet.
Add complete protease inhibitors to sonication buffer chilled on ice. Six plates yield around 200 microliters of nuclear pellet Resus. Suspend the pellet in 15 pellet volumes or three milliliters of the sonication buffer.
Using a different volume for this amount of cells might change the sonication efficiency. Sonication efficiency should be determined beforehand for your lab's ator. Here a Miss Sonic sonicate 3000 equipped with the microchip is used.
Sonicate the nuclei suspension on ice for 10 cycles for 20 seconds on and 40 seconds off at an amplitude setting of eight. This typically results in chromatin fragments averaging 200 to 1000 base pairs in size when sonication is complete. Transfer the sonicated material to an SS 34 centrifuge tube and centrifuge at 13, 000 RPM for 20 minutes of four degrees Celsius.
In an SS 34 rotor to pellet cell debris, which is usually a small amount. Carefully transfer the supinate to a 15 milliliter conical tube on ice. Proceed to chip to decrease background from non-specific binding events.
First, pre-clear the chromatin preparation by adding 360 microliters of salmon sperm DNA protein A or G agro bead slurry and rocking for four degrees Celsius for at least 30 minutes. Centrifuge at 1000 RPM for five minutes at four degrees Celsius to pellet the agro beads. Then transfer the pre-cleared supine natin to another 15 milliliter conal tube on ice.
Next, measure the absorbance of 260 nanometers or the A 260 units of the chromatin. Dilute 10 microliters of preclude chromatin in 190 microliters of double distilled water. For the blank, combine 10 microliters of sonication buffer with 190 microliters of double distilled water.
Adjust the volume of the sample with sonication buffer to four A 260 units. Save 75 microliters of the pre-cleared chromatin at four a two 60 units as the input sample and store at minus 20 degrees Celsius until ready to reverse cross links. Remember that this step is crucial for data analysis.
So do not neglect to take the input aliquot dispense the remaining chromatin preparation in one milliliter aliquots into 1.7 milliliter micro refuse tubes on ice. If stopping here, snap. Freeze the prepared chromatin on dry ice and store at minus 80 degrees Celsius.
However, since stored chromatin can degrade, it is best to continue with the ip. Now add the chip grade antibodies to the induced and un induced sample sets. In duplicate.
Typically one to two micrograms of antibody works well, but the amount must be determined empirically beforehand. Add two micrograms of IgG and 15 microliters of pan histone H three antibody as the negative and positive controls to induced and un induced sample sets. As well rock all tubes overnight at four degrees Celsius before proceeding to isolate the immuno complexes following the overnight incubation with the antibodies.
Add 60 microliters of salmon sperm DNA protein A or protein giro bead slurry to all tubes. Rocket four degrees Celsius for at least 60 minutes. To bind the immuno complexes to the beads.
Pellet the immuno complexes bound to the agros beads by micro gently at 2000 RPM for three minutes at four degrees Celsius aspirate the supernatant. Next perform a series of buffer washes, starting with a low salt wash followed by a high salt wash. Then lithium chloride wash.
And finally, two TE washes to each of the wash buffers. Add PMSF just prior to use wash with one milliliter of buffer for 10 minutes with rocking at four degrees Celsius micro for three minutes at 2000 RPM at four degrees Celsius and aspirate the supernatant keeping the samples chilled on ice. After removing the final tea buffer wash, add 500 microliters of freshly prepared Lucian buffer to the beads vortex to mix the aros beads in the Lucian buffer and heat for 30 minutes at 65 degrees Celsius.
Vortexing every 10 minutes while the samples are eluting, thaw the input samples and add 500 microliters of elution buffer to each one. Include these samples in all steps going forward. Once the samples finish the 65 degree Celsius incubation micro fusion per 30 seconds at 2000 RPM and transfer the EENT to a new micro fuge tube.
Finally, reverse the cross-linking. Add five molar sodium chloride to a final concentration of 200 millimolar to all samples and vortex incubator 65 degrees Celsius for at least four hours up to overnight. Once the cross links of the immuno complexes are reversed, the bound DNA can be recovered when the incubation at 65 degrees Celsius is complete.
Micro fuge samples for 30 seconds. To collect condensation on lids, add one microliter of RNAs A to each tube and incubate room temperature for 15 minutes following the RNAs treatment to each tube at 10 microliters of 0.5 molar EDTA 40 microliters of 0.5 molo tris chloride pH 6.5 and two microliters of proteinase K or a total of 52 microliters from a master mix and incubate at 45 degrees Celsius for 60 minutes. Extract the samples with phenol chloroform, iso aile alcohol, and then with chloroform ISO A my alcohol.
Then to precipitate the DNA, add two microliters of glycogen and 0.1 volumes of three molar sodium acetate. pH 5.2 to each sample and vortex, add 2.5 volumes of 100%ethanol vortex and incubate at minus 20 degrees Celsius overnight. Retrieve the samples and micro refuse at 13, 000 RPM for 20 minutes at four degrees Celsius.
Wash the DNA glycogen pellets with one milliliter of 70%ethanol and air. Dry the pellets or use a speed vacuum centrifuge. Do not over dry the DNA re suspend the pellets in 200 microliters of TE buffer and quantify no more than one microliter by real time.
PCR shown here are the results of a chip experiment following the dynamics of H three K 36 ME three. During interferon gamma induction of the IRF one gene H three K 36 ME three antibody was used to pull down chromatin collected from two FTGH cells induced with interferon gamma for 30 minutes, 1.5 hours and five hours with IgG as a negative control. The occupancy of STAT one was also determined at these time points as was RNA polymerase two occupancy.
The histone occupancy across the IRF one gene locus was determined for the un induced and induced conditions using the pan histone three antibody, which also serves as a positive control. The dip around minus five base pairs is due to the nucleosome depletion that is found at transcription start sites. We've just shown you the procedure for chromatin immunoprecipitation.
This assay is useful for observing chromatin modifications that occur during activated gene transcription. When doing this procedure, it is important to work out the shearing procedure for your chromatin with your ator before starting a chip experiment. Also remember to take your input samples.
So that's it. Thanks for watching and good luck with your experiments.
