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Genome-wide Chromatin Maps Derived from Limited Numbers of Hematopoietic Progenitors

ABSTRACT: Whole genome mapping of protein-DNA interactions can be performed by coupling chromatin immunoprecipitation (ChIP) with high-throughput sequencing (ChIP-Seq). However, current methods require large amounts of starting materials which precludes their application to rare cell types. Here, we combine a high-sensitivity ChIP assay with a novel library preparation procedure to map histone modifications in as few as 10,000 cells. We apply the technique to acquire genome-wide chromatin maps for an enriched population of hematopoietic progenitors, and thereby gain insight into their developmental program.

ORGANISM(S): Mus musculus

PROVIDER: GSE22075 | GEO | 2010/07/01

SECONDARY ACCESSION(S): PRJNA128955

REPOSITORIES: GEO

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Genome-wide Chromatin Maps Derived from Limited Numbers of Hematopoietic Progenitors

Project description:Whole genome mapping of protein-DNA interactions can be performed by coupling chromatin immunoprecipitation (ChIP) with high-throughput sequencing (ChIP-Seq). However, current methods require large amounts of starting materials which precludes their application to rare cell types. Here, we combine a high-sensitivity ChIP assay with a novel library preparation procedure to map histone modifications in as few as 10,000 cells. We apply the technique to acquire genome-wide chromatin maps for an enriched population of hematopoietic progenitors, and thereby gain insight into their developmental program. An optimized ChIP protocol for small number of cells and a novel Librray preperation methods for high throughput sequencing of picograms amount of ChIP DNA.

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Project description:Previously, we introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing. The scale itself was detemined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. In this paper we extend those results in several ways. First, we simplified the calculations defining the quantitative scale. Second, we highlight the normalization constraint implied by the quantitative scale and introduce a new scheme for generating 'tracks' for siQ-ChIP. We next introduce some whole-genome analyses that are unique to siQ-ChIP which allow us, for example, to project the IP mass onto the genome to evaluate how much of any genomic interval was captured in the IP. We apply these analyses to p300/CBP inhibition and demonstrate that response to inhibition is a function of genomic architecture. In particular, active transcription start sites are only weakly perturbed by p300/CBP inhibition while enhancers are strongly perturbed. Similar observations have been reported in the literature, but without a quantitative scale, those observations have been misinterpreted. We discuss how the siQ-ChIP approach precludes such misinterpretations, which stem from the wide spread community practice of treating unquantified and unnormalized ChIP-seq tracks as though they are quantitative.

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Project description:Current methods for whole-genome mapping of protein-DNA interactions, performed by coupling chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), require large amounts of starting materials, which precludes their application to rare cell types. Here we combine a high-sensitivity ChIP assay with a new library preparation procedure to map histone modifications in as few as 10,000 cells. We used the technique to characterize mouse hematopoietic progenitors and thereby gain insight into their developmental program.

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