Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles

M. Mokry, P. Hatzis, E. de Bruijn, J. Koster, R. Versteeg, J. Schuijers, M.L. van de Wetering, V. Guryev, H. Clevers, E. Cuppen

Onderzoeksoutput: Bijdrage aan wetenschappelijk tijdschrift/periodieke uitgaveArtikelWetenschappelijkpeer review

Samenvatting

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.
Originele taal-2Engels
Pagina's (van-tot)15092
TijdschriftPLoS One
Volume5
Nummer van het tijdschrift11
DOI's
StatusGepubliceerd - 2010

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