The overall goal of this procedure is to prepare chromatin from adult mouse skeletal muscle. This method can help answer key questions in gene regulation and skeletal muscle fibers. The main advantage of this technique is that it allows preparation of ChIP-grade chromatin from mouse skeletal muscle.
A physically resistant tissue with a high content of structural proteins. We first had the idea of this method when we were seeking to characterize the molecular mechanisms underlying gluco-articulated use muscle atrophy, which requires identification of gluco-articulated receptor, DNA-binding sites in mouse skeletal muscle fibers. Demonstrating the procedure will be Shilpy Joshi, a post-doc from our lab.
To begin this procedure, recover frozen or freshly dissected hindlimb muscles. Subsequently, mince the muscles in a two-milliliter test tube, containing one milliliter of ice cold hypotonic buffer to a homogeneous preparation of small pieces, using fine scissors. Afterward, leave the tube shaking on a bench top agitator at four degrees Celsius for five to 10 minutes.
Next, transfer the dissected tissue to a 14-milliliter round-bottom tube containing four milliliters of cold hypotonic buffer. Homogenize the minced tissue, using a mechanical tissue homogenizer for 15 to 30 seconds. Afterward, transfer the homogenate to a 15-milliliter tube.
Add cold hypotonic buffer to result in 10 milliliters of the sample and fix the homogenate before proceeding with the next mouse. In this procedure, add formaldehyde to the sample to result in 1%final concentration. And then shake for 10 minutes at room temperature.
Next, add glycine to result in a final concentration of 0.125 molar in each tube to stop fixation. And shake for five to 10 minutes at room temperature. After that, homogenize the fixed lysate, using a loose dounce.
Then, transfer it to a 15-milliliter tube and centrifuge at 1000 g for five minutes at four degrees Celsius to obtain nuclei and cellular debris. After five minutes, remove the supernatant and re-suspend the pellet in five milliliters of fresh hypotonic buffer. Filter the lysate through a 70-micrometer cell strainer into a 50-milliliter tube.
Using a pipette tip, wash the filter with one milliliter of cold hypotonic buffer. Following that, refilter the filtrate through a 40-micrometer cell strainer. Subsequently, transfer the filtrate to a 15-milliliter tube.
And centrifuge at 1000 g for five minutes at four degrees Celsius to obtain the nuclear pellet. After discarding the supernatant, use one milliliter of hypotonic buffer to pool the three pellets and transfer to a 1.5 to two-milliliter tube. Centrifuge at 1000 g for five minutes.
Next, assess the volume of the nuclear pellet by comparison with tubes containing a known volume of liquid and re-suspend it in sonication buffer up to three to four times of packed nuclear volume. Subsequently, sonicate the suspension for 10 to 15 minutes at four degrees Celsius, using a sonicator. Centrifuge the sonicated sample and transfer the chromatin supernatant to a fresh 1.5-milliliter tube.
To de-crosslink, take 30 microliters of chromatin in a 1.5-milliliter test tube and add 20 microliters of five molar sodium chloride and 450 microliters of water. Complete the volume to 500 microliters and incubate at 65 degrees Celsius overnight. The next day, perform a treatment with one microliter of Proteinase K, 10 microliters of two molar tris at pH 6.8 and 10 microliters of 0.5 molar EDTA for one hour at 42 degrees Celsius.
Then, perform a regular phenol-chloroform chloroform extraction and precipitate the DNA with one volume of sodium acetate and two volumes of 100%ethanol for one hour at negative 80 degrees Celsius or overnight at negative 20 degrees Celsius. Next, pellet the DNA by centrifugation at 13, 500 g for 15 minutes. Decant the supernatant and wash the pellet thoroughly with cold 70%ethanol and centrifuge again for five minutes.
Then decant the supernatant and air-dry the pellet. After that re-suspend the pellet in the original volume of tris HCl EDTA buffer. Measure the DNA concentration in a spectrophotometer by absorbents at 260 nanometers, 280 nanometers.
Subsequently, pipet 500 to 1000 nanograms of DNA together with a DNA-sized ladder on separate lanes of a 1.5%agarose gel. And perform electrophoresis to assess the fragment size of chromatin. To optimize, homogenization was performed for either 15 seconds or 45 seconds at two speeds, 18, 000 and 22, 000 RPM.
In all conditions, nuclei could be separated from the tissue debris, but the yield was optimal using lower speed. Nuclei could also be prepared by douncing for three to five minutes. But mechanical homogenization is the method of choice as optimal yields of nuclei can be achieved in as little as 15 seconds, allowing an important gain of time, especially if multiple samples have to be processed.
Using this protocol, up to 2.7 times 10 to the seven nuclei from 500 milligrams of tissue can be isolated to generate 100 micrograms of chromatin. Chromatin prepared in this way gives clean, high-quality results in ChIP sequencing experiments. As illustrated by the high levels of acetylated K27 of histone H3 and RNA polymerase 2 at the muscle-expressed Desmond locus.
Specificity for muscle fiber is shown by low or absent signal at the Pecam1 and Vcam1 loci that are not expressed in muscle. While attempting this procedure, it is important to properly immerse the homogenizer probe in the sample to avoid frothing, to clean the probe between samples, and to optimize the chromatin fragmentation. Chromatin prepared in this way has been successfully used for ChIP's sake to map genome-wide transcription factor occupancy and should also be suitable for chromatin confirmation capture techniques.
After it's development, this technique paved the way for research in the field of genomics, to explore gene regulation in skeletal muscle from mice. The procedure can also be applied to muscle from rat and biopsies from human patients. After watching this video, you should have a good understanding of how to use mechanical homogenization, followed by purification of formaldehyde-fixed nuclei to prepare chromatin from skeletal muscle for immunoprecipitation.
Don't forget that working with formaldehyde, phenol, chloroform, and Proteinase K can be hazardous. Precautions such as wearing of gloves and glasses should be taken. Steps involving these reagents should be performed under a hood.
