title: Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles.
abstract: 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.
authors.affiliation: Cancer Genomics Center, Department of Medical Genetics, Hubrecht Institute and University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Michal
authors.initials: M
authors.lastname: Mokry
authors.forename: Pantelis
authors.initials: P
authors.lastname: Hatzis
authors.forename: Ewart
authors.initials: E
authors.lastname: de Bruijn
authors.forename: Jan
authors.initials: J
authors.lastname: Koster
authors.forename: Rogier
authors.initials: R
authors.lastname: Versteeg
authors.forename: Jurian
authors.initials: J
authors.lastname: Schuijers
authors.forename: Marc
authors.initials: M
authors.lastname: van de Wetering
authors.forename: Victor
authors.initials: V
authors.lastname: Guryev
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
mesh_terms: D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002843:Chromatin; D004268:DNA-Binding Proteins; D009711:Nucleotides; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D004247:DNA