Hi,
I've just started doing ChIP-seq, and am having difficulty getting my INPUT and IP DNA fragment sizes in the proper size range for Illumina sequencing. My ChIP protocol seems to work fine, as I can show enrichment of a 360bp region surrounding a known binding site in my IP sample compared to the INPUT sample by pcr.
However, our Bioanalyzer reports that the size fragmentation is too small for the library prep. This seems to indicate that I'm over-sonicating the samples. I've been following a protocol published earlier this year by Mike Snyder's lab http://dx.doi.org/10.1016/S0076-6879(10)70004-5. In this protocol the sonication procedure uses a Branson Digital Sonifier @ 5x30sec @ power 50.
In our lab, we have a Branson Analog Sonifier and I've tried sonicating 6x10sec @ power 20 and 3x10sec @ power 20. In both cases, the bioanalyzer indicates that my DNA size fragmentation is mostly <100bp. However, when I run the INPUT DNA on a 2% Agarose gel, I see a smear with the greatest DNA concentration ~1,000bp and no DNA below 100bp.
I've just started doing ChIP-seq, and am having difficulty getting my INPUT and IP DNA fragment sizes in the proper size range for Illumina sequencing. My ChIP protocol seems to work fine, as I can show enrichment of a 360bp region surrounding a known binding site in my IP sample compared to the INPUT sample by pcr.
However, our Bioanalyzer reports that the size fragmentation is too small for the library prep. This seems to indicate that I'm over-sonicating the samples. I've been following a protocol published earlier this year by Mike Snyder's lab http://dx.doi.org/10.1016/S0076-6879(10)70004-5. In this protocol the sonication procedure uses a Branson Digital Sonifier @ 5x30sec @ power 50.
In our lab, we have a Branson Analog Sonifier and I've tried sonicating 6x10sec @ power 20 and 3x10sec @ power 20. In both cases, the bioanalyzer indicates that my DNA size fragmentation is mostly <100bp. However, when I run the INPUT DNA on a 2% Agarose gel, I see a smear with the greatest DNA concentration ~1,000bp and no DNA below 100bp.
Comment