This method can help answer key questions in the epigenetics field such as how much chromatic modifications are regulated in a cell type specific manner along with genomes. The main advantage of this technique is that we can isolate chromatin from the targeted cells and then follow typical gypsy downstream. So this method can provide insights into neuron specific trimincil, lysinc 4, opisoncilly.
It can also be applied to other histone modification, other cell types, and then other model organisms. Demonstrating the procedure will be Mari Mito, a technician from my laboratory. To begin, use fine spring scissors to dissect the tissue of interest into small pieces.
Add the tissue fragments to a clean container filled with liquid nitrogen. Then, transfer the tissue fragments to two milliliter tubes filled with liquid nitrogen. Store the tubes at minus 80 degrees Celsius for five minutes with the cap open to evaporate the liquid nitrogen.
Place the tubes containing the tissue samples on a chilled metal ice rack. Then, place a metal bullet in a 1.5 milliliter tube and chill it with liquid nitrogen. Place the chilled metal bullet to one of the sample tubes.
Close the cap and set the tube into the tube holder of a cryogenic grinder. Immediately, dip the assembled tube holder in liquid nitrogen for one minute. Insert the frozen tube holder into the outer cassette of the cryogenic grinder.
And shake it vigorously for 30 seconds, 60 times. After this, disassemble the cryogenic grinder, remove the metal bullet. And place the tube into a pre-chilled sample cooler.
Store the cooler at negative 20 degrees Celsius for 15 minutes. Then, add 900 microliters of 1%formaldehyde to the tube and pipette to mix. Transfer the suspension to a new two milliliter tube with 900 microliters of 1%formaldehyde.
Next, fix the suspension for 10 minutes with gentle rotation at 23 degrees Celsius. To stop the fixation reaction, add 100 microliters of 2.5 molar glycine to the tube. And centrifuge at 3, 000 times gravity for five minutes at four degrees Celsius.
After this, discard the super supernatant. Add one milliliter of PBS to the tube and vortex to mix. Centrifuge at 3, 000 times gravity for five minutes at four degrees Celsius.
Repeat this wash step, two more times. Next, add 500 microliters of lysis buffer one to the pellet and pipette to mix. Centrifuge the tube for five minutes at 3, 000 times gravity at four degrees Celsius and discard the supernatant.
Add one milliliter of lysis buffer two to the pellet and vortex to mix. Then, centrifuge the suspension for five minutes at 3, 000 times gravity at four degrees Celsius and discard the supernatant. After this, add 800 microliters of radio immunoprecipitation asssay buffer with protease inhibitor cocktail to the pellet and pipette to mix.
Centrifuge the suspension for five minutes at 3, 000 times gravity at four degrees Celsius and discard the supernatant. Next, add 500 microliters of RIPA buffer with protease inhibitor cocktail to the pellet. Then, centrifuge the suspension for five minutes at 3, 000 times gravity at four degrees Celsius and discard the supernatant.
Add one milliliter of RIPA buffer with protease inhibitor cocktail. Immediately after adding RIPA buffer with protease inhibitor cocktail, transfer the lysate to a sonicator tube. And place the tube on an ultrasonicator.
Using the settings outlined in the text protocol, shear the chromatin. Then, transfer the sample to a 1.5 milliliter protein low binding tube. And centrifuge it for five minutes at 20, 000 times gravity at four degrees Celsius.
Collect the supernatant from the sample and transfer it to a new protein low binding tube. In this protocol, tandem chromatin immunoprecipitation sequencing was introduced and demonstrated. During the procedure, the quality of the DNA was tested at multiple steps.
Immediately after sonication, the sheared DNA was isolated and tested with microfluidic electrophoresis machine. After using anti-FLAG antibody and anti-H3K3me3 antibody to perform affinity purification the DNA quality was checked again. The specificity of the immune purification of DNA on H2B-FLAG was confirmed with negative control samples.
Where negligible amounts of DNA were detected. Further into the protocol, the quality of the sequencing library was verified. The successful ChIP and T-ChIP were evidenced via enrichment analysis using primers targeting the promoter region of the GAPDH gene.
Representative redistributions along three neuron genes were obtained. And the enrichment of reads at the five prime ends of the genes by neuron T-ChIP seek over whole brain T-ChIP seek was observed. After a stabiliment, this technique paves the way for researchers in the epigenetic field to explore cell type specific histone modification general widely in neurons in mice.
Don't forget that working with formaldehyde can be extremely hazardous and to take precautions such as wearing rubber clothing and glasses should always be taken while performing this procedure.
