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I am standardizing the shearing of mouse brain tissue using the covaris. For the sample I am using tissue to extract nuclei and then FACS to get specific nuclei of interest followed by chromatin shearing. Covaris gives a specific protocol to use based on the starting material and the specific use of either milliTube or microTube. My question is that even though I am starting with a specific amount of tissue in grams, following flow I am left with a small number of cells ~400k does it change the if i shear my chromatin in 1ml for millitube or 130ul for microtube.
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Hi Shldbcrzy1,
can you please provide a few details of your workflow:
1. are you cryofrcturing the fixed tissue as advised for tissue ChIP?
2. what starting mass ranges of tissue are you working with?
3. Since you are working with FACS sorted nuclei, and working with only 400K cell equivalents of nuclei, you should shear in the 130ul volume in a shearing buffer containing 1mM tris, 10mM EDTA, ph 7.6, 0.1% SDS. after shearing you can then add the missing components to match that of the constituents of your IP buffer. After the missing components are added to the sheared chromatin, clear the lysate and carry out the IP. Shearing 400k cells in larger volume will simply dilute the chromatin which will then require a larger amount of antibody for the IP step.
if you have further questions, please contact [email protected]
Thank you.
hamid
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