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Internally Calibrated ChIP-seq (ICeChIP-seq) using a large panel of antibodies against H3K4 methylations to measure genome-wide histone modification density in mammalian cells

ABSTRACT: Internally Calibrated ChIP-seq (ICeChIP-seq) using a large panel of antibodies against H3K4 methylations to measure genome-wide histone modification density in mammalian cells

PROVIDER: PRJNA401866 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12684564_1.fastq.gz	Fastqsanger.gz
	SRR12684564_2.fastq.gz	Fastqsanger.gz
	SRR6010170_1.fastq.gz	Fastqsanger.gz
	SRR6010170_2.fastq.gz	Fastqsanger.gz
	SRR6010180_1.fastq.gz	Fastqsanger.gz

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Internally Calibrated ChIP-seq (ICeChIP-seq) using a large panel of antibodies against H3K4 methylations to measure genome-wide histone modification density in mammalian cells

Project description:Post-translational modifications (PTMs) on histone proteins regulate genome accessibility and are frequently studied using chromatin immunoprecipitation (ChIP). In ChIP, an antibody putatively specific towards a histone PTM is used to map its genomic locations. ChIP experiments assume perfect antibody-epitope specificity, an assumption previously shown to be problematic, largely through peptide array studies. Among the most well-studied histone PTMs are the mono-, di-, and tri-methylation states of histone H3 lysine 4 (H3K4). While each state has been ascribed different biological functions, the methylation state specificity of antibodies used in these studies has not been systematically interrogated. Here, we use internally calibrated ChIP (ICeChIP) to comprehensively define the specificities of 52 commercially available antibodies marketed to distinguish the three methylation states of H3K4, allowing identification of both high- and low-specificity antibodies. We then conduct ICeChIP-seq with 18 such antibodies of varying specificity. We further find that the sum of H3K4me1 and H3K4me2 across enhancers contacting a promoter correlates strongly with gene expression for all genes, including housekeeping genes, and note that use of low-quality antibodies yields materially different biological interpretations. These results illustrate the way by which variable specificity of commercial antibodies contributes to the “reproducibility crisis” in biological research and demonstrates the need to carefully validate antibodies with techniques appropriate for the intended applications.

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