sonication?

[jazz]

Hi guys!
I am new to the ChIP-seq technique, and this forum.I have a basic question about chromatin size. I have done a chromatin IP several times and have been successful in getting a shear size between 100-500bp using sonication. But I have heard that one has to achieve a much tighter distribution (+/- 50 bp) for a ChIP-seq! Is it true? How does one get that? Do you prefer MNase digestion, or is it OK if I just gel purify the band/area of interest?
Any suggestions would be helpful.

Thanks
-Jazz

[Chipper]

Yes, the tighter distribution is selected by gel purifiction.

[fang]

Hi,Chipper! Thank you for your suggestion!
I just met the same problem in Chip-seq and even worse. I only got 500-1000bp by sonication.
Could you please tell us more details? Can we excise the bands and extract with the Qiagen gel extraction kit?
Thanks a lot!

[sem]

We have been successfully using the protocol outlined by Illumina for size selection of ~200bp fragments. http://www.rockefeller.edu/genomics/...ample_Prep.pdf
Good Luck

[Chipper]

fang,
what conditions / sonicator are you using? Perhaps you need to titrate the sonication conditions (number of cycles, cell concentrations etc).

[fang]

Thank you Sem!
I have down loaded the protocol from illumina.
but my question is the chromatin DNA fragments used for ChIP cann't be sheared smaller by sonication. So I consider to excise the chromatin DNA by gel extraction.Do you think it is feasible? Thanks!

[fang]

Chipper,
I use Misonix ultrasonic processor, XL2020.My condition is work on 15'', work off 20'', cycles from 15,20,25 to 30. I use embryonic stem cells for 1.5*107 in 300ul lysis buffer. I always get smear focusing in 500-1000bp, no smaller. If I add the power, it is in emulsification and I'm afraid that the protein and DNA may be denatured.

Could you please give me some advices? Thanks

[fang]

Chipper,
I have one more question. How do you think of enzyme digestion the genomic DNA instead of sonication? I'm planning to try enzymatic shearing. Any suggestion is helpful!
Thanks a lot!

[sem]

fang - I don't know if I understand your question. What I can say is that we use the Bioruptor (in the cold room) on high for 30 sec on/60 sec off for 10 minutes. I do this twice, placing more ice in the water bath before the second run. Our gels to examine fragment size always show smears rather than a nice clean band. We have tried to address this without much success. However, we have continued with the material and it seems fine. We have performed many experiments (ChIP-qpcr, ChIP-chip, and ChIP-seq) with various controls and it is ok.
After the ChIP, you will size select the material for 200 bp pieces prior to the amplification required for the sequencing.
Also, maybe you have too much material in your sample when you sonicate. Try an experiment where you do several dilutions in the same conditions and volume and see if that makes a difference.
MNase is ok for certain things but if you are IPing for transcription factors, you are better off with sonication.
Good Luck!

[fang]

Thank you very much, Sem! Your suggestioin will be greatly helpful!
Following your experience, I will try it again and expect to get ideal results!
Yes, I'm going to do some ChIP-seq on transcription factor, so sonication is exactly very important. I expect more discusion with you if possible.
How do you quantity the DNA before Seq with illumina? Nanodrop or Qubit fluorometer, and the sequence center in our school recomends the latter?
Thanks a lot!

[sem]

fang - After the amplification, we use the Bioanalyzer to check the size of the fragments as well as the concentration.

[huguesparri]

Fang:
In our lab', we are using Qubit to quantify our sample concentration before we start the Illumina's ChIP-Seq sample prep. protocole.
As the amount of starting material is at a low concentration, Qubit appears to be (in our experience) more accurate than nanodrop or Agilent Bioanalyzer.

On the other hand, we use the Agilent Bioanalyzer at the end of Illumina's protocole in order to validate the library: it gives a rather accurate estimation of the library concentration and size wich is necessary to calculate the amount of material (=number of molecules) we are going to introduce in the Cluster Station.

[fang]

Thank you Sem!

Thank you Sem! I'll go on my work. I hope to discuss with you if I have problems. Thanks!

[scientist]

problem

Hello,
I have a problem like this: I am using Bioruptor from Diagenode and trying to shear chromatin from peripheral blood mononuclear cells. I want to get pieces from 500 bp. We use 1% SDS lysis buffer, I tryed all different cycles,but my sonication does not show results. Every time I put it on gel, I get too big chromatin pieces.
I hope someone will help me.
Thank you very much...

[captainentropy]

scientist, my lab also uses the Bioruptor. A while back we (and the other lab that uses it) noticed a dropoff in the sonication ability of the machine. The aluminum foil test was done and the results indicated we needed servicing. After the unit was serviced the sonication was back to normal, even better actually. So, perhaps your Bioruptor needs servicing.