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X-linked ChIP protocols

August 16, 2011

Here are four ChIP protocols.

ChIP (MNase fragmentation) protocol

ChIP (fragmentation in 0.1% SDS – tip sonicator) protocol

ChIP (fragmentation in 1% SDS – tip sonicator) protocol

ChIP (fragmentation with Covaris) protocol

The first protocol uses micrococcal nuclease to shear the DNA and seems to produce excellent results in some situations. This is based on an Epigenome NOE protocol (http://www.epigenome-noe.net/WWW/researchtools/protocol.php?protid=10) but I find the modifications I made substantially increase the yield of chromatin. The second uses a tip sonicator with sonication in a buffer containing 0.1% SDS.  This is based on a protocol from the Myers Lab (http://myers.hudsonalpha.org/documents/Myers%20Lab%20ChIP-seq%20Protocol%20v041610.pdf). The third protocol uses a tip sonicator with sonication in buffer containing 1% SDS and is based on the Farnham ChIP protocol.  The fourth protocol uses the Covaris instrument to shear the DNA with a 1% SDS shearing buffer. This was a protocol from Covaris that I found on their website although there is no link to it on their website (huh??).  It’s partially from the Vivek Mittal Lab – Yes the Volley Lama himself (fellow alumni of that epic Hernandez Downtown Lab volleyball team the Invisible Pellets).

All the protocols have some advantages/disadvantages.  One of the biggest considerations is the SDS concentration.  For the protocol using 1% SDS you have to dilute your samples 1:10 to reduce the SDS concentration to 0.1% before you do your IP.  This can result in dilute samples and if you need a lot of DNA back, as you do for ChIP-seq, it can cause some difficulties.  However, 1% SDS greatly increases the sonication efficiency.  I have found it very difficult shear DNA down to the size for ChIP-seq using 0.1% SDS.  The amount of time sonicating is a little more than my brain can physically endure.  Really, a few hours sonicating samples is enough to give you the mother of all headaches.  I think the choice really comes down to enzymatic shearing or using the Covaris instrument (assuming you have access to one) and what is best entirely depends on the epitope.

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