Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding ProfilesReportar como inadecuado




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Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing NGS procedures. Because these larger fragments may be non-random and represent relevant biology that may otherwise be missed, but also because they represent a significant fraction of the immunoprecipitated material, we designed a double-fragmentation ChIP-seq procedure. After conventional crosslinking and immunoprecipitation, chromatin is de-crosslinked and sheared a second time to concentrate fragments in the optimal size range for NGS. Besides the benefits of increased chromatin yields, the procedure also eliminates a laborious size-selection step. We show that the double-fragmentation ChIP-seq approach allows for the generation of biologically relevant genome-wide protein-DNA binding profiles from sub-nanogram amounts of TCF7L2-TCF4, TBP and H3K4me3 immunoprecipitated material. Although optimized for the AB-SOLiD platform, the same approach may be applied to other platforms.



Autor: Michal Mokry, Pantelis Hatzis, Ewart de Bruijn, Jan Koster, Rogier Versteeg, Jurian Schuijers, Marc van de Wetering, Victor Gurye

Fuente: http://plos.srce.hr/



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