Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

The overall goal of this procedure is to identify the enrichment of histone marks in a specific genomic location. This method can help answer key questions in the epigenetics field, such as how mutations in epigenetic regulators which alter histone modifications impact the tumor cells transcriptome. The main advantage of this technique is that cells are not cross-linked, so they are in a more natural state, which can aid antibody specificity.

Begin by dissecting the experimentally induced tumor from the brain of a mouse in a 10 centimeter Petri dish. If the tumor expresses a fluorescent protein, visualize the tumor through a fluorescent dissecting microscope set to the appropriate fluorescent channel in 0.3 times magnification. Use a scalpel and forceps to separate the tumor tissue from the normal brain, and mince the tumor into small pieces in the Petri dish.

Transfer the dissected tumor to a 1.5 milliliter tube with 300 microlitres of NSC medium, and use a disposable syringe to gently homogenize the tumor. Then, add one milliliter of cell detachment medium, and incubate the sample for five minutes at 37 degrees Celsius. After the incubation, pass the cell suspension through a 70 micron cell strainer into a 50 milliliter centrifuge tube.

Then, wash the strainer with 25 milliliters of NSC medium and centrifuge the suspension at 300 times G for five minutes at room temperature. After decanting the supernatant, re-suspend the pellet in six milliliters of NSC medium and transfer the tumor cell suspension into a T25 culture flask. Culture the cells at 37 degrees Celsius in a tissue culture incubator with an atmosphere of 95%air and 5%carbon dioxide until neurospheres are formed.

Once the neurospheres are harvested, centrifuge one times 10 to the six neurosphere cells per amino precipitation at 300 times G for five minutes. After centrifuging, decant the supernatant and re-suspend the neurosphere pellet in one milliliter of balanced salt solution. Transfer the suspension to low protein binding 1.5 milliliter micro centrifuge tubes then centrifuge as before.

Next, decant the supernatant and re-suspend the cell pellet in 95 microliters of digestion buffer, supplemented one to 100 with protease inhibitor cocktail per one times 10 the the six cells. Immediately pipette the cells up and down to prevent clumping and avoid creating any bubbles. Then, mix five microliters of 0.1X micrococcal nuclease in 145 microliters of digestion buffer and add five microliters of diluted micrococcal nuclease per one times 10 to the six cells.

Flick the tube to mix and place the tube in a 37 degree Celsius heat block for exactly 12 minutes. The micrococcal nuclease digestion to fragment chromatin is the most critical step. Proper chromatin fragmentation is important for achieving a high resolution for ChIP.

To ensure the step is successful, incubate multiple samples one at a time to ensure over-digestion does not occur. After 12 minutes, add 10 microliters of 10X micrococcal nuclease stop buffer per one times 10 to the six cells. Samples must be kept on ice from this point onwards.

Bioanalyzer analysis of the micrococcal nuclease digested sample demonstrates that most of the DNA has been fragmented into mono, di, and tri-nucleosomes. Next, add 110 microliters of 2X ripa buffer supplemented one to 100 with protease inhibitor cocktail per one times 10 to the six cells. Flick the tube to mix, then centrifuge for 15 minutes at four degrees Celsius in 1700 times G.Transfer the supernatant which contains the chromatin to a regular 1.5 milliliter micro centrifuge tube, and place on ice.

Use ripa buffer supplemented with protease inhibitors at one to 1000 to dilute the chromatin, so that each IP has a volume of 100 to 200 microliters. Then, remove an aliquot equivalent to 10%of the ChIP volume as the input sample. Prepare the input by adding 100 microliters of TE buffer supplemented with proteinase K, and incubate at 55 degrees Celsius for one hour.

After purifying the input with a PCR purification kit, measure the concentration of the input using a micro volume spectrophotometer. Record the results in a notebook. Add 10 microliters of washed protein A and G magnetic beads per IP that will be performed on each sample, and incubate for one hour at 4 degrees Celsius with rotation at 20 RPM.

Place the sample on a magnetic holder and allow the beads to separate, then transfer the volume required into new 1.5 milliliter tubes. Add the desired antibody to each tube and then wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 degrees Celsius with rotation as before.

The next day, spin the tubes at 2000 times G for 10 seconds, then add 10 microliters of beads to each IP and incubate for 3 hours at 4 degrees Celsius with rotation at 20 RPM. Then, place the samples on a magnetic stand and allow the magnetic beads to separate. Use a pipette to remove the supernatant, then add 150 microliters of ripa buffer with protease inhibitors to each IP and incubate at 4 degrees Celsius at 20 RPM for five minutes.

After the incubation period, place the samples on a magnetic stand and allow the magnetic beads to separate. Use a pipette to remove the supernatant, then add 150 microliters of lithium chloride buffer supplemented with protease inhibitors at low concentration to each IP, and incubate at 4 degrees Celsius with rotation at 20 RPM for five minutes. After the incubation period, place the samples on a magnetic stand and allow the magnetic beads to separate, then use a pipette to remove the supernatant.

Then, add 150 microliters of cold TE without protease inhibitors to the beads, and incubate at 4 degrees Celsius with rotation at 20 RPM for five minutes. After the final washing step, re-suspend sample in 100 microliters of TE buffer supplemented with the final concentration of 0.5 milligrams per milliliter proteinase K, and incubate at 55 degrees Celsius for one hour. Finally, purify the amino precipitated DNA using a PCR purification kit.

Then, to check if the IP is successful, perform a qPCR with primers targeted to positive and negative control regions. qPCR was performed with IGG ChIP DNA, histone-3-lycene-4-trimethylated ChIP DNA and histone-3-lycene-27-thrimethylated ChIP DNA, using primers for glyceraldehyde-3-phosphate-dehydrogenase. Representative results demonstrate that Gapdh, a housekeeping gene, is only enriched with the H3K4me3 modification associated with active transcription, and not the H3K27me3 modification associated with repressed chromatin.

Following this procedure, other methods like ChIP-C can be performed in order to identify changes in histone marks, genome-wide. After watching this video, you should have a good understanding of how to perform native ChIP in neurospheres to identify histone mark at specific gene sites.