I and my colleagues are trying to master the methodology of chromatin immunoprecipitation (ChIP) of mouse skeletal muscle. We performed ChIP protocol and used sonication to shear the chromatin to ~200-1000bp. Gel electrophoresis was used to separate sheared DNA fragments by size. Gel images taken after 20, 40 and 60 minutes are shown below. As you can see, they drastically differs from images taken after successful sonication. For example, there are no differences between control and sheared (sonicated) samples. We would be very grateful for any information, comments and suggestions how to solve this problem. The deeper explanation of performed procedures are shown below.
ChIP protocol
We removed the tibialis anterior muscle from mouse, weighted on balance and placed to the tube (1.5 mL). We washed muscle with PBS and removed it. After that added ice-cold PBS (10µL PBS per milligram of tissue) and chopped fresh muscle into small pieces using scissors for 2 minutes at a comfortable rate on ice. To crosslink chromatin and proteins we removed tube from ice and added formaldehyde to a final concentration of 1 % and everything swirled gently on a platform at room temperature for 15 minutes. The cross-linking was stopped by adding glycine to a final concentration of 0.125 M and swirled gently for 5 minutes. Then we centrifuged muscle sample for 5 minutes, 720 rpm, 4 °C and removed supernatant. Thereafter pellets were washed with ice-cold PBS (1mg tissue - 10µL PBS), centrifuged for 5 minutes, 720 rpm, 4 °C and removed supernatant. This procedure was repeated 2 times. In order to distinguish nucleus from we homogenized sample on ice in 1x PBS (50/100 mg muscle) and everything centrifuged for 10min, 1000rpm, 4 °C. Supernatant was removed and pellet was suspended in Lysis Buffer (750 µL).
Sonication was performed under these conditions:
Amplitude 20% from max (500 watts), Samples were sonicated 4 times for 10sec. After each sonication 5 µL of sample was removed. During sonication samples were kept on the ice all the time. After sonication samples were centrifuged for 10 min, 12000 x g at 4 °C and stored at fridge (-81 °C) for 3 days.
In order to degrade RNA and detach proteins from sheared chromatin we:
treated samples with RNase A and incubated them at 37 °C for 0.5 hour. Thereafter samples were treated with proteinase K and were incubated at 37 °C for 2 hours. Lastly, samples were used in gel electrophoresis (parameters: 2% agarose gel, 60V) to separate sheared DNA fragments by size.
Please to not hesitate to ask, if more specific information is required.
Thank you.
You need to heat sample to 65oC for at least 4 hours to reverse the chemical crosslinks. Some protocols say boiling for 10 minutes in high pH buffer.
My protocol as follows:
1) Each of tissue was minced to≈1 mm-sized piece, and immediately was crosslinked with 1% formaldehyde (1ml per 100 mg tissue) for 15 min at room temperature. Then crosslinking reaction was stopped by adding glycine to a final concentration of 0.125M.
2) The sample was placed on ice, washed six times with 1 ml cold PBS containing proteinase inhibitors and then frozen at – 70 ° C for 5 min. The sample was vortexed and spin down at 200g at 4°C for 10 s before and after wash.
3) The Chromatin was solubilized and extracted in a cell lysis buffer by homogenized twice for 10 sec.
4)The homogenate was centrifuged at 5500 * g for 5 min.
5)The supernatant, containing extracellular debris, was decanted, and the pellet was homogenized two times, for 10 sec, using nuclear lysis buffer.
6)The extracted chromatin was sheared to 200- 1000 bp using the Sonicator. Each sample was sonicated fourteen times on ice, 20 sec each, at 70% of maximum power. During sonication, the Chromatin sample was cooled on ice
Optimization of DNA Shearing
The desired fragment size should be determined prior to the immunoprecipitation (IP) by performing various sonication conditions on isolated Chromatin and the sonication efficiency was evaluated by gel electrophoresis using the procedure below.
1. 20 μl of sonicated Chromatin was added to 100 μl of PCR-grad water.
2. The 100 µl of mixed DNA purifying slurry was added to the sonicated Chromatin/ H2O mix.
3. The samples were mixed by inversion and incubate for 10 min, 98°C.
4. After incubation, the samples were leaved at room temperature for 20 min to cool.
5. The samples were centrifuged briefly for 10 sec, 10000 x g to remove condensation from the tube lid.
6. 1 µl of proteinase K was added, vortexed for 5 sec at medium power.
7. The samples were incubated firstly at 55°C for 30 min and then at 98°C for 10 min.
8. DNA purifying slurry was pelleted by centrifugation, 14 000 x g, for 1 min at room temperature.
9. The supernatant was transferred to a 1.5 ml tube without disturbing the DNA purifying slurry pellet.
10. 10 µl of extracted DNA was loaded on a 1.5 % agarose gel to analyze its fragment size
be careful During sonication, the Chromatin sample was cooled on ice and donot form bubble
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