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Old 03-02-2014, 02:56 AM   #1
joshuab
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Location: australia

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Default covaris sonication for ChIRPseq

Hi,

I am planning to do ChIRPseq which requires sonication of chromatin down to around a 100-500bp range. The protocol recommends using a Bioruptor and suggests that it may take up to 4 hours for sonication to that level. We have access to a Covaris M220 but not a Bioruptor so I wanted to try the sonication using the Covaris machine.

One critical point is that the aim of the protocol is to pull down RNA which is bound to the chromatin so any sonication process needs to minimize RNA damage. We would also like to examine any proteins pulled down (the process uses biotin labelled oligos which bind to the RNA) probably using mass spectrometry so need the proteins in reasonable condition also.

The initial cross linking is done using glutaraldehyde which I believe requires a longer sonication than formaldehyde treated cells.

Any advice regarding the use of Covaris vs Bioruptor or suggested alternative protocols would be greatly appreciated.

Thanks

Joshua
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Old 03-04-2014, 10:44 PM   #2
richi17
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Location: Hungary

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Hi ,

Check this out..

Diagenode-Bioruptor Pico

Last edited by richi17; 03-05-2014 at 12:05 AM. Reason: additional info
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Old 12-17-2014, 07:18 AM   #3
TeRible
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with the Bioruptor Pico you can shear for 8 cylces (30 sec ON, 30 sec OFF) chromatin (fixed with 1% formaldehyde) and get fragments in the range 100-500 bp. It is mild to proteins, I don't know about RNA but in general formaldehyde crosslinks nucleic acids to protein, so if the RNA is simultaneously binding to DNA and protein it should also be crosslinked.
Check this paper for some tips 'The genomic binding sites of a noncoding RNA' by Matthew D.Simon 2011
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