The overall goal of this procedure is to analyze epigenetic modifications during brain development, especially in the cerebral cortex and the cerebellum. This method can be used to answer key questions in the neurobiology field, such as how histone modifications like H3K79 methylation can affect the development of cortical and cerebellar layering. The main advantage of this technique is that we can study in vivo the histone modifications, locus-specific or genome-wide, in distinct brain regions at specific developmental time points.
Begin with brains from P5 to P7 and MRI mice. Remove all meninges and blood vessels. Then transfer three to five cerebella per chip into 15-milliliter tubes.
Wash cerebellar with HBSS glucose before centrifugation. Centrifuge the tubes to collect the tissue. After the final wash, homogenize the cerebella by gently pipetting up and down two to three times with a one-milliliter pipette until the fragments are 0.5 to one-cubed millimeters in size.
Gently remove all liquid until 2.5 milliliters are left. Then add 0.05%trypsin into the tube with HBSS glucose containing the cerebella. And incubate the tissue in a 37 degrees Celsius water bath for 15 minutes.
Invert the tissue every one to three minutes. Following the incubation, stop the digestion by adding five milliliters of CGNP cell culture medium, or CGM, and collect the tissue by centrifugation as before. After centrifuging, remove the supernatant and add one milliliter CGM.
Triturate the tissue with a one-milliliter pipette tip, avoiding the formation of air bubbles. Then add five milliliters of CGM and incubate the mixture for two minutes on ice to settle tissue remnants. Once the tissue has settled, transfer the supernatant into a new 15-milliliter tube.
Then add two milliliters of CGM to the residual tissue and repeat the trituration procedure before harvesting the supernatant and discarding the tissue remnants. Pool the supernatants from each 15-milliliter tube of tissue and centrifuge to collect the cerebellar cells. Resuspend the pellet in 10 milliliters of CGM.
Since astrocytes adhere faster and stronger to poly-D-lysine than CGNPs, add up to four milliliters of cell suspension to the wells of poly-D-lysine-coated 6-well plates and incubate for 20 minutes at 37 degrees Celsius to remove astrocytes. Shake the plate, collect the supernatant in a 15-milliliter tube and then centrifuge the supernatant as before. Resuspend the pellet in 10 milliliters of CGM and count the cells with a Neubauer counting chamber.
After four to six hours, the adhered CGNPs appear round and are proliferative. Seed the cells in CGM on poly-L-ornithine-coated plates and incubate at 37 degrees Celsius, 5%CO2 and 100%relative humidity. After one day in vitro, or DIV1, most CGNPs are round and proliferate.
Some CGNPs will start to differentiate and begin outgrowth of neuronal protrusions. To fix the cultured cells, add up to one milliliter of 1%PFA directly to the wells containing CGNPs. After a five-minute incubation at 22 degrees Celsius, add 53 microliters of glycine and wash twice with three milliliter of ice-cold PBS.
Use a cell scraper to harvest the cells in one milliliter of PBS. Transfer the cells into 1.5-milliliter tubes and centrifuge for five minutes at 1, 000 g and four degrees Celsius. After removing the supernatant, add 700 microliters of lysis buffer containing protease inhibitor and incubate the samples for 15 minutes at four degrees Celsius.
Vortex the samples every five minutes during this time. Following the incubation, divide the sample tubes to ensure that the volume does not exceed 400 microliters per tube. Then sonicate for 10 minutes at maximum power to shear the chromatin of the lyse cells.
After 10 minutes, vortex the samples and then repeat the sonication and the vortexing two more times. Check the lysate for remaining nuclei with a phase contrast microscope or fluorescent microscope using DAPI. Pellet the cell remnants by centrifuging for 10 minutes at 13, 000 g and four degrees Celsius.
When finished, use the supernatant for the preclearing step. To preclear the samples, first wash 20 micro liters of unconjugated magnetic beads per sample in ice-cold PBS Tween. Remove the washes with aid of a magnetic stand.
Then add 600 microliters of dilution buffer and 20 microliters of washed magnetic beads to each sample for preclearing and incubate for two hours at four degrees Celsius on a rotator. Following the incubation, remove the beads using a magnetic stand. Then take 5%of each lysate as input samples and freeze them at minus 20 degrees Celsius.
Antibody-coupling can be performed while the samples are being precleared. Wash 45 microliters of Protein A Magnetic Beads per chip with ice-cold PBS Tween, as before. After the final wash, add one milliliter of ice-cold PBS to the beads.
Then add three micrograms of antibody per 45 microliter aliquot of beads. Incubate for two hours at four degrees Celsius on a rotator to bind the antibodies to the beads. When the incubation time has elapsed, resuspend washed antibody-coupled beads in one bead volume of ice-cold PBS.
Divide each precleared extract into two tubes of approximately 643.5 microliters. Add the same volume of dilution buffer and antibody-bound beads and incubate the samples at four degrees Celsius overnight on a rotator. The next day, wash the beads with ice-cold chip buffers one through three for 10 minutes each on a rotator at four degrees Celsius, follow with three five-minute washes with TE buffer.
Add elution buffer and dilute for one hour at 1, 400 RPM in a shaker at room temperature. Once the hour has elapsed, add 10 micrograms of Rnase to each chip sample and five micrograms to the input samples. Incubate the samples on the shaker for 30 minutes at 37 degrees Celsius.
Finally, add 100 micrograms of Proteinase K per chip sample and 50 micrograms per input. Incubate overnight at 65 degrees Celsius at 1, 400 RPM. H3K79 dimethylation levels are decreased after three days of CPC culturing and DOT1L inhibition by SGC 0946.
QCPR after chip analysis revealed that genes with high histone-3-lysine-79 methylation levels at baseline were most responsive to the inhibitor treatment. Genes with a lower H3K79 dimethylation coverage showed no significant change in methylation levels. This heat map shows that histone-3-lysine-79 methylation levels peak around the transcription start site and decrease toward the transcription end site.
Once mastered, this technique can be done in about one week, if it is performed properly. After watching this video, you should have a good understanding in how to perform chromatin immunoprecipitation on histone modifications in isolated brain cells.
