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Old 10-07-2013, 02:51 PM   #1
mamocol
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Location: La Jolla, CA

Join Date: Oct 2013
Posts: 1
Default ChIP-exo: shearing bacterial genomic DNA using Covaris S220

Hello everyone,

I'm trying to optimize sonication for ChIP-exo for better reproducibility. I have never utilized the Covaris S220 system and cannot find a protocol for bacterial DNA shearing for ChIP. Here are my thoughts

I am shearing E. coli and am targeting 250 bp fragments. Before sonication, I have crosslinked with formaldehyde and lysed with lysozyme. The settings I am thinking of using are the following:

Duty Cycle: 10%
Intensity: 5
Cycles per Burst: 200
Time: 130 seconds

Following sonication, I plan on performing a phenol chloroform extraction followed by Nucleospin columns and running a Bioanalyzer to determine fragment sizes. The purpose of this experiment is to optimize the sonication settings to get more reproducible data.

Any comments, advice, or protocols will be greatly appreciated.

Thanks!

-M
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Old 10-21-2013, 03:56 PM   #2
pmad
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Location: University of Cambridge, UK

Join Date: Jun 2010
Posts: 14
Arrow

Hi,
If you are producing or analyzing ChIP-exo data (Rhee HS, Pugh BF, 2011, Cell 147:1408-19) with replicates, you might want to consider CexoR to uncover high-resolution protein-DNA interactions. It processes data data in BAM aligment format.

http://bioconductor.org/packages/dev...tml/CexoR.html [1]
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Old 12-06-2013, 01:39 PM   #3
Anna Maria
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Location: Boston, MA

Join Date: Jan 2010
Posts: 2
Default Using Covaris S220 for ChIP-exo

Hi,
Shearing of bacterial crosslinked DNA for ChIP is very similar to shearing crosslinked chromatin from mamalian cells. Is even simpler since bacteria doesn't have the nuclei that are usually isolated before chromatin shearing is performed.

How was your sample fixed (type of formaldehyde, buffer composition, temperature)?
How many cells?
What is the composition of the buffer in which you want to shear your sample?
What is the sample volume?
What's size of the tube you are planning to use?

The AFA conditions you listed look like the protocol for the purified DNA shearing in S2/E210 instrumnets, since S220 doesn't have Intensity because this parameter was replaced by Peak Incident Power. Shearing of DNA with the crosslinked proteins requires different conditions.

We are using two types of tubes (microTUBES for 130ul samples) and milliTUBES for 1 ml samples) and conditions for chromatin shearing in mammalian cells in those tubes are provided in the protocol on our web site http://covarisinc.com/wp-content/uploads/pn_400103.pdf.
However, these conditions are for the buffers we are providing in the kit and the chromatin shearing is performed in a simple buffer consiting of Tris, EDTA and 0.1% SDS. When using a different buffer this conditions can be used as a starting point and will require further optimiztion. You will need to perform a time course to find the right lenght of the treatmnet time.

If you provide more details I might be able to provide you with a better recommendations.

Anna
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bacterial dna, chip seq, covaris, sheared dna

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