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Sonication woes for Chip-seq, using Bioruptor - (Jun/16/2012 )

Hello Everyone,
Before post here, I have searched the forum with "sonication" or "Bioruptor", but my woes still exist. I'm using the Bioruptor UCD-200 to sonicate the cultured MCF-7 or other cancer cells for chip-seq. I have tried 30-60 cycles at low power or 15-40 cycles at high power. Although sometimes i got the ideal fragments (200-500bp),the results were very inconsistent. What do you all think of the results that I am getting (see attached images)? I am using the same fixation as well for 1% formaldehyde and then glycine to stop the fixation at RT.The cells were lysated with Cell Lysis buffer (20 mM Tris-HCl pH8, 85mM KCl, 0.5% NP-40, 15min), and followed by Nuclear Lysis buffer (50mM Tris-HCl pH8, 10 mM EDTA, 1%SDS,30 min). As advised in some paper,a snap-frozen step was added before sonication. other conditions were detailed in images. I use the 1.5-mL Eppendorf tube and the concentration is about 5*10^5 cells/120 uL/tube. By the way, according to the Bioruptor manual, I spin down and vortex every 10 cycles. But when spin down, there would be white precipitates at the bottom of the tubes.Is the precipitate SDS? In addition, I just leave very little ice in the tank since it would decrease the sonication efficiency. So, what is the deal?
Thanks a lot for any input.
Attached Image

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-jancho.wang-

Hey Jancho,

Unfortunately, we were having a very similar problem with the BioRuptor. Sometimes, it would work very well (i.e. <500 bp fragments) and then other times, it wouldn't work at all (i.e. huge smearing). We tried many different solutions, but in the end, wound up having to use another lab's Covaris E210 machine.

That being said, maybe some of the things that we tried could be helpful to you, given that we used a different cell line:

-For our sonications, we tried using different buffers--1% SDS (as you are doing), 0.1% SDS, and a buffer without SDS (with sodium deoxycholate and N-lauroyl sarcosine). When using the 1% SDS buffer, we limited the amount of time the samples were on ice. We would add the buffer then go to the BioRuptor without putting the samples on ice. In addition, a friend had suggested gently warming up the tube between each sonication step (by rubbing the sides of the tube) to try to decrease the chance of SDS precipitation.

-I agree about the problem with the ice decreasing the sonication efficiency. In fact, although I'm not positive, it's even possible that slight differences in the amount of ice (however little) could contribute to the variability that you are observing. Our BioRuptor is in a cold room, so that decreases the need for a large amount of ice. Right before using the BioRuptor, we added ice and mixed it until it melted. The water bath usually hovered around 2-4ºC. We tried not adding any more ice to the machine and, after 5 minutes at low power, we tested the temperature and it never really got above 10-11ºC. I know that the manual says that the temperature should not exceed 10ºC, but it's at least worth trying out to see so that you don't accidently add different amounts of ice.

-I'm sure that almost everyone that will respond to this post will suggest using the more thin-walled tubes for sonicating, specifically the TPX tubes as opposed to Eppendorf tubes. From our experience, when the sonication worked, the shearing looked MUCH better using the TPX tubes vs. normal Eppendorf tubes. Again, we could not always reproduce this....

We tried to deal with our BioRuptor problem for about 2-2.5 months, but we simply were unable to get consistent shearing. I sincerely hope that you are able to solve your problem.

-yanks1ny-

yanks1ny on Sat Jun 16 16:06:58 2012 said:


Hey Jancho,

Unfortunately, we were having a very similar problem with the BioRuptor. Sometimes, it would work very well (i.e. <500 bp fragments) and then other times, it wouldn't work at all (i.e. huge smearing). We tried many different solutions, but in the end, wound up having to use another lab's Covaris E210 machine.

That being said, maybe some of the things that we tried could be helpful to you, given that we used a different cell line:

-For our sonications, we tried using different buffers--1% SDS (as you are doing), 0.1% SDS, and a buffer without SDS (with sodium deoxycholate and N-lauroyl sarcosine). When using the 1% SDS buffer, we limited the amount of time the samples were on ice. We would add the buffer then go to the BioRuptor without putting the samples on ice. In addition, a friend had suggested gently warming up the tube between each sonication step (by rubbing the sides of the tube) to try to decrease the chance of SDS precipitation.

-I agree about the problem with the ice decreasing the sonication efficiency. In fact, although I'm not positive, it's even possible that slight differences in the amount of ice (however little) could contribute to the variability that you are observing. Our BioRuptor is in a cold room, so that decreases the need for a large amount of ice. Right before using the BioRuptor, we added ice and mixed it until it melted. The water bath usually hovered around 2-4ºC. We tried not adding any more ice to the machine and, after 5 minutes at low power, we tested the temperature and it never really got above 10-11ºC. I know that the manual says that the temperature should not exceed 10ºC, but it's at least worth trying out to see so that you don't accidently add different amounts of ice.

-I'm sure that almost everyone that will respond to this post will suggest using the more thin-walled tubes for sonicating, specifically the TPX tubes as opposed to Eppendorf tubes. From our experience, when the sonication worked, the shearing looked MUCH better using the TPX tubes vs. normal Eppendorf tubes. Again, we could not always reproduce this....

We tried to deal with our BioRuptor problem for about 2-2.5 months, but we simply were unable to get consistent shearing. I sincerely hope that you are able to solve your problem.

Thanks a lot for your respond.
I'll try to use TPX tubes and remove all the ice in later sonication. SDS precipitation maybe a serious problem. During sonicating, the precipitation at the bottom would affect the ultrasound from the tank bottom, wouldn't it? Gently warming up the tube may be a choice. How did others deal with the problem?

-jancho.wang-

As mentioned by yanks1ny , TPX tubes improve significantly the shearing. For chromatin shearing, always use High Power. Using the cooling system from Diagenode instead of ice could help with consistency and reproducibility. Do you performed the reverse of cross-linking ( the pictures look like no de-crosslinking were perform)? The fixation may also affect the shearing efficiency. The best way is to trypsinize the cell and fix them in suspension in PBS but on directly on dishes. Fixing of dishes might results in the formation of cell aggregates which are difficult to disrupt. You can find a lot of tips on chromatin shearing with the Bioruptor at http://www.diagenode.com/media/documents/downloads/protocols/The_Ultimate_Guide_for_Chromatin_Shearing_Optimization_with_Bioruptor_protocol.pdf.

-epigeneticfan-

The problem is the SDS, not the tubes..............Run your nuclear pellet through a 20G syringe 10 times in the SDS buffer then place your tubes in the 4degree until your ready to sonicate..........don't leave them in the ice bucket for any extended period of time unitl after the sonication.

-chabraha-

Triton-X-100 prevents SDS from precipitation.

-memari-