Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq
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ABSTRACT:
PROVIDER: PRJNA287213 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR2065050.fastq.gz | Fastqsanger.gz | |||
| SRR2065060.fastq.gz | Fastqsanger.gz | |||
| SRR2065070.fastq.gz | Fastqsanger.gz | |||
| SRR2065080.fastq.gz | Fastqsanger.gz | |||
| SRR2065090.fastq.gz | Fastqsanger.gz |
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Project description:Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein’s distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C.glabrata cells (the calibration genome) with S.cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this “internal standard” calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using PCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.
2015-07-10 | GSE69907 | GEO
Project description:Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein’s distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C.glabrata cells (the calibration genome) with S.cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this “internal standard” calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using PCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics. Develop a method for quantitative ChIP-seq
2015-07-10 | E-GEOD-69907 | biostudies-arrayexpress
Project description:Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.
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Project description:Abf1 has been defined as a general regulatory factor involved in determining chromatin structure in the yeast Saccharomyces cerevisiae. As such, it plays a major role in a range of functions including DNA replication, transcriptional activation and gene silencing, as well as DNA repair. Previous studies have identified an Abf1 DNA consensus binding sequence and Abf1 binding at up to several hundred locations throughout the genome. Depletion of Abf1 in the cell, however, results in altered nucleosome structure at many thousands of sites throughout the yeast genome, suggesting a far greater role for Abf1 in chromatin structure and dynamics. Here, we examine genome-wide Abf1 binding using ChIP-Chip to measure its chromatin occupancy. Using a novel software package, Sandcastle, we detect 3,821 genomic positions at which Abf1 binds. We conducted a detailed bioinformatic analysis of these novel Abf1 binding sites, defining variations in the current consensus sequence and identifying many more genomic locations at which Abf1 can be found. These observations define the sites at which Abf1 occupancy determines chromatin structure, providing a framework for understanding how processes such as replication, transcription and DNA repair are organised within the genome.
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