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Hi everyone:
I posted this same thing on another forum but thought it most useful here (sorry for the double post)
I've been lurking here for awhile and have finally decided to post. I have read many of the threads on generation of ChIP-seq libraries and have gained considerable knowledge about the technique. I have been doing ChIP for about 8-10 years using a homemade protocol (actually from the Farnham lab) and has worked very well for all the applications I have tried. I use Staph A cells (Pansorbin) to pull down the antibody/chromatin complexes and by qPCR get very clean results. I mostly study 1-2 transcription factors and have very good ChIP antibodies. I have also generated a considerable amount of ChIP-chip data. Now I'm ready to move to ChIP-seq. For these libraries, I actually switched to using a magnetic Protein A bead kit for the ChIP because I was told that there may be carry over Staph A DNA in the library and that I should size select larger fragments (500-600bp) to avoid carrying over these sequences. The Mag kit (Diagenode) seemed to work well so I proceeded to make my first libraries. I quantitated the ChIP product by Qubit and started with 20 ng TI DNA and ChIP DNA. I used the Illunina kit with a 1:30 dilution of the adaptors and did 18 cycles of PCR. I size selected after ligation and again after PCR using E-gels. I have plenty of product. I did qPCR for a few known binding sites and got mixed results. For one a couple of the targets the enrichment looks great but for one there doesn't seem to be enrichment. I know that if my PCR primers are not exactly over the binding site or if the amplicons are a bit long I can get strange results. I'm having my libraries run on a bioanalyzer to check for purity (no adaptor complexes or primer dimers). If everything looks good I plan to send for sequencing.
Thanks for reading this far. I was wondering if anyone else uses Staph A cells for ChIP and have made libraries that have been successfully sequenced. I like the new Mag bead kit but it is a lot more expensive than my homemade protocol.
Cheers,
Karen
I posted this same thing on another forum but thought it most useful here (sorry for the double post)
I've been lurking here for awhile and have finally decided to post. I have read many of the threads on generation of ChIP-seq libraries and have gained considerable knowledge about the technique. I have been doing ChIP for about 8-10 years using a homemade protocol (actually from the Farnham lab) and has worked very well for all the applications I have tried. I use Staph A cells (Pansorbin) to pull down the antibody/chromatin complexes and by qPCR get very clean results. I mostly study 1-2 transcription factors and have very good ChIP antibodies. I have also generated a considerable amount of ChIP-chip data. Now I'm ready to move to ChIP-seq. For these libraries, I actually switched to using a magnetic Protein A bead kit for the ChIP because I was told that there may be carry over Staph A DNA in the library and that I should size select larger fragments (500-600bp) to avoid carrying over these sequences. The Mag kit (Diagenode) seemed to work well so I proceeded to make my first libraries. I quantitated the ChIP product by Qubit and started with 20 ng TI DNA and ChIP DNA. I used the Illunina kit with a 1:30 dilution of the adaptors and did 18 cycles of PCR. I size selected after ligation and again after PCR using E-gels. I have plenty of product. I did qPCR for a few known binding sites and got mixed results. For one a couple of the targets the enrichment looks great but for one there doesn't seem to be enrichment. I know that if my PCR primers are not exactly over the binding site or if the amplicons are a bit long I can get strange results. I'm having my libraries run on a bioanalyzer to check for purity (no adaptor complexes or primer dimers). If everything looks good I plan to send for sequencing.
Thanks for reading this far. I was wondering if anyone else uses Staph A cells for ChIP and have made libraries that have been successfully sequenced. I like the new Mag bead kit but it is a lot more expensive than my homemade protocol.
Cheers,
Karen