This protocol results in reproducible and reliable chromatin extraction from frozen liver specimens for chromatin immunoprecipitation experiments. To begin, place the tube containing the frozen tissue in the mortar, and let it sit there for five minutes. Then apply pressure to the sample using a pre-chilled pestle until there are no more solid crumbles.
Remove the tube containing the sample from the mortar. Add 950 microliters of ice cold PBS with the required inhibitors, and pipette up and down gently until the sample is completely resuspended. Immediately transfer the tissue suspension to the homogenizer, and apply 20 to 30 strokes with Pestle A to obtain a finer suspension.
Avoid moving the pestle outside of the liquid phase to prevent foaming. Transfer the homogenate to a new 1.5 milliliter tube previously chilled on ice. Then centrifuge the tube for five minutes at 1, 300 G and four degrees Celsius.
Carefully remove the supernatant and resuspend the pellet completely in 950 microliters of room temperature PBS by gentle pipetting. Then add 63.6 microliters of 16%methanol-free formaldehyde to obtain a final concentration of 1%Immediately rotate the tube for 10 minutes at room temperature. After rotation, add 113 microliters of 1.25 molar glycine at room temperature to obtain a 125 millimolar final concentration, and rotate the tube for another five minutes.
Then centrifuge the sample at 1, 300 G for three minutes at four degrees Celsius. Discard the supernatant, and resuspend pellet carefully by pipetting in 950 microliters of ice cold PBS with the required inhibitors. Centrifuge the sample again and resuspend the pellet as previously demonstrated.
Repeat the centrifugation step once more and immediately proceed to the chromatin isolation procedure. Add 950 microliters of Buffer A with the required inhibitors to the pellet and mix gently by pipetting until the pellet is completely resuspended. Then rotate the tube for 10 minutes at four degrees celsius.
After rotation, centrifuge the sample at 2, 000 G for five minutes at four degrees Celsius and carefully remove the supernatant. Add 950 microliters of Buffer B with the required inhibitors to the pellet and mix gently by pipetting until the pellet is completely resuspended. Then rotate the tube for 10 minutes at four degrees celsius.
After rotation, centrifuge the sample again. Remove the supernatant. Then add 300 microliters of room temperature Buffer C with the required inhibitors to the pellet and pipette vigorously.
Vortex the sample for 15 to 30 seconds. Then spin the tube briefly to collect the drops on the lid. After sonicating the chromatin sample, add 30 microliters of 10%Triton X-100 solution and vortex for five to 10 seconds.
Centrifuge the sample at 16, 000 G for 15 minutes at four degrees Celsius. And transfer the supernatant to a clean 1.5 milliliter tube pre-chilled on ice. Transfer 10 to 25 microliters of sheared chromatin to a new tube, and add Buffer C to reach a final volume of 200 microliters.
Aliquot and shock freeze the rest of the chromatin at minus 80 degrees Celsius until further use. Add eight microliters of five molar sodium chloride and incubate for at least six hours at 65 degrees Celsius in the heating block while shaking at 1, 000 RPM. Extend the incubation overnight if possible.
After letting the samples cool at room temperature for five minutes, add two microliters of RNase A and incubate for one hour at 37 degrees Celsius while shaking at 1, 000 RPM. Remove the samples from the heating block and add seven microliters of 300 millimolar calcium chloride and two microliters of proteinase K.Incubate the samples in the heating block at 56 degrees Celsius for 30 minutes while shaking at 1, 000 RPM. Meanwhile, prepare one phase separation tube for every sample by centrifuging them down to 16, 000 G for one minute at four degrees Celsius.
After removing the tubes from the heating block and letting them equilibrate at room temperature for three minutes, transfer 400 microliters of the sample to the previously centrifuged phase separation tube. Next, add 400 microliters of phenol chloroform isoamyl alcohol solution, and vortex for five seconds. Centrifuge the tube at 16, 000 G for five minutes at four degrees Celsius.
Then add 400 microliters of chloroform and vortex for five seconds. Centrifuge the tube for another five minutes and transfer 400 microliters of the upper phase to a new 1.5 milliliter tube that contains 24 microliters of five molar sodium chloride and 0.75 microliters glycogen. Then briefly vortex the tube.
Add 1, 055 microliters of 100%ethanol. Then vortex thoroughly and incubate the sample at minus 80 degrees Celsius for one hour or at minus 20 degrees Celsius overnight. After the incubation is complete, centrifuge the sample at 16, 000 G for 30 minutes at four degrees Celsius and carefully remove the supernatant without dislocating the pellet.
Add 500 microliters of cold 70%ethanol and tilt the tube gently to ensure the pellet is washed. Centrifuge the tube at 16, 000 G for 15 minutes at four degrees Celsius. Carefully remove the whole supernatant and let the pellet dry at room temperature.
Alternatively, incubate the tube at 37 degrees Celsius for quicker drying. Add 50 microliters of Tris-EDTA solution and put the tube on the heating block for five to 10 minutes under shaking at 300 RPM. Then analyze the DNA on a 1%agarose gel.
After decross-linking of chromatin and visualization of the DNA on agarose gel, successful shearing can be recognized by the presence of fragments in the range of 100 to 300 base pairs. The chromatin was successfully precipitated with H3K4 trimethylation, H3K27 acetylation, and H3 K27 trimethylation antibodies and further analyzed by qPCR. Chromosome one open reading frame 43, proteasome 20S subunit beta two, and glyceraldehyde-3-phosphate dehydrogenase promoter regions that are actively transcribed in the liver showed enrichment for H3K4 trimethylation and H3K27 acetylation.
In comparison, homeobox C13, homeobox C12, and the mouse myelin transcription factor one promoter regions that are silenced in the liver were not enriched as expected. H3 K27 trimethylation shows the opposite behavior, thereby confirming the success of the chromatin immunoprecipitation or ChIP assay. The chromatin was successfully subjected to ChIP-Seq.
After sequencing, the reeds were aligned to a previously prepared index and separated according to species. H3K4 trimethylation and H3K27 acetylation deposition at gene transcription starting sites antagonized with H3K27 trimethylation deposition as expected. The Hox C cluster is known for being transcriptionally inactive in both mouse and human livers.
The profiling of H3K4 trimethylation and H3K27 acetylation shows peaks for these two post-translational modifications outside of the cluster, while the signal intensity of H3K27 trimethylation tends to increase within the gene cluster. Chromatin extraction from frozen tissue specimens has high intrinsic variability, strongly depending on cell suspension fineness and temperature during cross-linking. Ensuring fixed laboratory conditions helps in maintaining fragment size range reproducibility.
