The overall goal of this experimental procedure is to allow preparation of high-quality nuclei from cross-linked skeletal muscle which can then be used for chromatin immunoprecipitation studies. This method can help answer key questions in muscle and circadian field such as genomic studies using muscle tissues and cells in circadian or other time-sensitive context. The main advantage of this technique is that it allow immediate cross-linking of samples and the filtration-based procedure can be reliably conducted to isolate high-quality nuclei with minimal myofibril contamination.
Although this method can provide insight into skeletal muscle, it can also be applied to other systems such as cardiac and smooth muscles. Weigh and mince hind limb skeletal muscle isolated from approximately 20-week-old C57 black six male mice in ice cold phosphate-buffered saline as described previously. Place the minced skeletal muscle tissue in a 50 milliliter conical tube containing 10 milliliters of PBS on ice.
Then centrifuge the sample at 300 times g at four degrees Celsius for five minutes. Carefully remove the PBS supernatant by aspiration. Estimate the pellet volume in each 50 milliliter tube by using reference tubes with one, two, three, and four milliliters of water.
Add seven volumes of ice cold 1%formaldehyde and PBS and homogenize the samples on ice using a probe tissue homogenizer. Next, cross-link the sample by incubating it at room temperature for 10 minutes. Quench the cross-linking reaction through the addition of one molar glycine to a final concentration of 0.125 molar followed by incubation for five minutes at room temperature.
After centrifuging the sample at 3, 000 times g for five minutes at four degrees Celsius, remove the supernatant via aspiration. Rinse the pellet by resuspending it in 10 milliliters of ice cold base buffer containing freshly added protease inhibitors. Centrifuge the sample again and carefully remove the supernatant by aspiration.
Then resuspend the pellet in six milliliters of lysis buffer containing freshly added protease inhibitors. Transfer the sample to a pre-chilled 15 milliliter Dounce homogenizer and incubate for 10 minutes on ice. Dounce homogenize each sample on ice with 15 slow strokes using a loose pestle followed by 15 strokes with a tight pestle to release the nuclei.
The following filtration steps are the key to isolation of high-quality nuclei. Filter the homogenate through a cell strainer and rinse with four milliliters of lysis buffer. Then centrifuge the samples at 1, 000 times g for 10 minutes at four degrees Celsius.
Resuspend the pellet in five milliliters of ice cold base buffer and Dounce 10 times with a tight pestle to release the nuclei. Then filter the suspension through a cell strainer and rinse the tube with two milliliters of base buffer and filter again. Repeat the filtration with cell strainers of gradually reduced pore size.
Finally, centrifuge at 1, 000 times g at four degrees Celsius for 10 minutes. Remove the supernatant and resuspend the pellet in 500 microliters of base buffer before centrifuging again. Discard the supernatant and store the pellet at minus 80 degrees Celsius.
Thaw the samples in 500 microliters of SDS lysis buffer and resuspend with gentle pipetting. Transfer the nuclei DNA suspension to a glass vial on ice. Choosing the right sonication platform and condition is critical for chromatin preparation from isolated nuclei.
Run the sonication with focused bursts of ultrasonic acoustic energy from a disc-shaped transducer using the settings outlined in the text protocol. Transfer the sonicated chromatin to a 1.5 milliliter tube. Then centrifuge at 12, 000 times g for 15 minutes and transfer the supernatant to a new tube.
Next, transfer 50 microliters of the sonicated sample into a new 1.5 milliliter tube for reverse cross-linking overnight at 65 degrees Celsius. Harvesting of the DNA samples is described in the following section. Following evaluation of sonication and quantitation as described in the text protocol, aliquot the thawed chromatin to approximately 100 to 120 micrograms per tube.
Then dilute the chromatin one to 10 with immunoprecipitation buffer. Next, add 40 microliters of a 50%slurry of BSA pre-blocked protein HE agarose or IgY beads and incubate on a rotator for three hours at four degrees Celsius. After centrifuging the sample at 1, 000 times g for 10 minutes, carefully transfer the supernatant to new tubes.
Then add antibodies at one microgram of antibody per 25 to 100 micrograms of chromatin DNA. Rotate gently overnight at four degrees Celsius and approximately 15 rpm. Then add 10 microliters of BSA pre-blocked protein HE agarose or IgY beads and mix.
Rotate the bead suspension in the cold room for two hours. Next, wash the beads with RIPA buffer followed by high salt buffer, lithium chloride buffer, and Tris EDTA as detailed in the text protocol. Following washing, elute the DNA from the beads by first centrifuging at 1, 000 times g for one minute and carefully removing the supernatant.
Resuspend the beads with 50 microliters of elution buffer. Then incubate for 10 minutes at 65 degrees Celsius. Following incubation, centrifuge at 12, 000 times g for five minutes.
Remove the supernatant and add another 50 microliters of elution buffer before centrifuging once again at 12, 000 times g for five minutes. The final elution volume will be 100 microliters. To reverse cross-link and perform DNA elution, first incubate the eluted chromatin overnight at 65 degrees Celsius.
Then add one microliter of RNase A before incubating for 30 minutes at 37 degrees Celsius. Subsequently, incubate with eight microliters of 10 milligrams per milliliter protease K for 30 minutes at 55 degrees Celsius. Harvest the DNA by using a PCR cleanup kit with an elution volume of 50 microliters according to the manufacturer's protocol.
Finally, analyze the profiles by real-time QPCR as previously described. Sequential filtration effectively removed tissue debris. Representative images of samples after the initial filtration and after serial filtration are shown here for comparison.
Progressive chromatin shredding through 10 cycles of sonication is revealed through running an agarose gel. 10 cycles of sonication with the digested chromatin resulted in DNA of approximately 500 base pairs. Here, representative QPCR results for B males one chIP with mouse skeletal muscle samples collected at ZT6 and ZT18 are shown.
Analysis of the Dbp locations elements on the Dbp gene revealed that consistent with previous results binding peaks at around ZT6. Once mastered, preparation of nuclei can be done in five to six hours if it's performed properly. After watching this video, you should have a good understanding of how to use the filtration-based methods to purify high-quality nuclei and chromatin DNA from cross-linked muscle tissue.
While attempting this procedure, it's important to remember to maintain samples on ice or in a cold room and save sample aliquots for microscopic checking before and after the filtration steps. After its development, this technique paved the way for the researchers in the field of muscle and genomics to explore circadian or time-sensitive dynamics of chromatin-protein interaction in various muscle tissues. Following this procedure, other methods like chIP sequencing can be performed in order to answer additional questions like look for patterns of transcription factor binding in muscle tissues.
