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Calibrating ChIP-seq with nucleosomal internal standards to measure histone modification density genome-wide

ABSTRACT: Despite serving as a central experimental technique in epigenetics research, chromatin immunoprecipitation (ChIP) suffers from several serious drawbacks: it is a relative measurement untethered to any external scale that obviates fair comparison amongst experiments; it employs antibody reagents that have differing affinity and specificity for target epitopes, which are in turn variable in abundance; and it is frequently not reproducible. To address these problems, we developed internal standard calibrated ChIP (ICeChIP), a method of spiking a native chromatin sample with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and revealing a correlation between the apparent symmetry of H3K4me3 in promoter nucleosomes and gene expression. Direct in situ assessment of immunoprecipitation accommodates for a number of experimental pitfalls, and provides a critical examination of untested assumptions inherent in conventional ChIP. Examination of spiked-in semi-synthetic nucleosomes in ICeChIP-seq experiments performed for HEK293, mESC E14 and DM S2 cell line

ORGANISM(S): Mus musculus

SUBMITTER: Alexander Ruthenburg

PROVIDER: E-GEOD-60378 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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