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

TY - JOUR

T1 - Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles

AU - Mokry, M.

AU - Hatzis, P.

AU - de Bruijn, E.

AU - Koster, J.

AU - Versteeg, R.

AU - Schuijers, J.

AU - van de Wetering, M.L.

AU - Guryev, V.

AU - Clevers, H.

AU - Cuppen, E.

N1 - Reporting year: 2010

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pone.0015092

DO - 10.1371/journal.pone.0015092

M3 - Article

SN - 1932-6203

VL - 5

SP - 15092

JO - PLoS One

JF - PLoS One

IS - 11

ER -