Hi Nitsan,

You could try to block the sepharose beads with BSA instead. My understanding is that blocking the beads with salmon sperm DNA just get you sequencing reads contamination. I've seen protocols where agarose or sepharose beads are blocked with salmon sperm ssDNA. However, I do not know whether this could represent a problem during sequencing. Here is a protocol to block the sepharose beads with BSA: http://mcb.berkeley.edu/labs/koshland/Protocols/Reagents/beads.html

Thank you Miguel.

Hi Nitsan,

I have done all my ChIP-seq with salmon sperm blocked Agarose/Protein A beads (Millipore 16-157) which worked fine: the large majority of reads were unique and mapped back to the mouse genome. These were with antibodies for histone modifications though, which generally result in higher recovery. It could therefore be that antibodies against transcription factors may have more trouble getting good numbers of specific reads.

I tried the same with the Protein G beads from the same company, and here I did have a large amount of a-specific DNA in my final eluate, which I suspected to be ssDNA (and therefore I didn't send for sequencing).

Daan

Hi Nitsan,

We have achieved many good results in ChIP-seq with our iDeal ChIP-seq kit for transcription Factors, one of our best-seller. It uses magnetic beads but they are also saturated with BSA which doesn't interfer with sequencing. Also fixation is key mainly with sensitive TF or inducible TF.

Doen't hesitate to contact me if you have any question or need any help.

Geoffrey

https://www.diagenode.com/p/ideal-chip-seq-kit-for-transcription-factors-x24-24-rxns

Sorry to add a question to the this thread (but i had a similar question), I am using the sigma protein a sepharose (cat# P9424) for the my downstream ChipSeq. My question is how do you prepare the 50% slurry (it's preswollen and supplied in 20% ethanol)

Hi Nitsan,

Still are you using sepharose beads that blocked with salmon sperm for your ChIP? What is the result? I am using protein G agarose beads blocked with salmon sperm DNA. Have you any suggestion for me?

Thanks

If you are still interested, we use Polyvinylpyrrolidone (1 mg/ml) along with BSA to block sepharose beads for our locus-specific ChIP or ChIP-seq experiments. Use of PVP instead of salmon sperm will avoid any sequencing issues. You can see Guertin and Lis, PloS Genetics, 2010 for further information.