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Simplified ChIP-exo assays II

ABSTRACT: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.

ORGANISM(S): Mus musculus

PROVIDER: GSE114606 | GEO | 2018/08/17

REPOSITORIES: GEO

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Simplified ChIP-exo assays

Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.

2018-08-17 | GSE110681 | GEO

Simplified ChIP-exo assays

Project description:Simplified ChIP-exo assays

| PRJNA434276 | ENA

TELP, a sensitive and versatile library construction method for next-generation sequencing

Project description:Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.

2015-12-15 | GSE75966 | GEO

TELP, a sensitive and versatile library construction method for next-generation sequencing

2015-12-15 | E-GEOD-75966 | biostudies-arrayexpress

Simplified ChIP-exo assays II

Project description:Simplified ChIP-exo assays II

| PRJNA471904 | ENA

Data Exploration, Quality Control and Statistical Analysis of ChIP-exo/nexus Experiments

Project description:ChIP-exo/nexus experiments present modifications on the commonly used ChIP-seq protocol for high resolution mapping of transcription factor binding sites. Although many aspects of the ChIP-exo data analysis are similar to those of ChIP-seq, these high throughput experiments pose a number of unique quality control and analysis challenges. We develop a statistical quality control pipeline and accompanying R package, ChIPexoQual, to enable exploration and analysis of ChIP-exo and related experiments. ChIPexoQual evaluates a number of key issues including strand imbalance, library complexity, and signal enrichment of data. Assessment of these features are facilitated through diagnostic plots and summary statistics calculated over regions of the genome with varying levels of coverage. We evaluated our QC pipeline with both large collections of public ChIP-exo/nexus data and multiple, new ChIP-exo datasets from E. coli. ChIPexoQual analysis of these datasets resulted in guidelines for using these QC metrics across a wide range of sequencing depths and provided further insights for modelling ChIP-exo data. Finally, although ChIP-exo experiments have been compared to ChIP-seq experiments with single-end (SE) sequencing, we provide, for the first time, comparisons with paired-end (PE) ChIP-seq experiments. We illustrate that, at fixed sequencing depths, ChIP-exo provides higher sensitivity, specificity, and spatial resolution than PE ChIP-seq and both significantly outperform their SE ChIP-seq counterpart.

2017-03-01 | GSE84830 | GEO

Glucocorticoid receptor profiling in IMR90, K562 and U2OS (ChIP-exo)

Project description:To identify the sequences responsible for recruitment of Glucocorticoid receptor (GR) to individual loci, we performed ChIP-seq and ChIP-exo that combines chromatin immunoprecipitation with an exonuclease digestion step. We performed these experiments in three cell lines : IMR90 (ATTC:CCL-186), U2OS osteosarcoma cell lines, K562 (ATCC:CCL243), upon glucocorticoid treatment.

2015-02-25 | E-MTAB-2956 | biostudies-arrayexpress

SALP, a new NGS library construction method especially useful for the high-throughput characterization of chromatin openness states

Project description:Background: Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. Results: This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3ʹ overhang of 3 random nucleotides, which can be efficiently ligated to the 3′ end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 105 to 500 cells. Conclusions: This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.

2018-01-31 | GSE104162 | GEO

Glucocorticoid receptor profiling in IMR90,K562 and U2OS (ChIP-seq)

2015-02-25 | E-MTAB-2955 | biostudies-arrayexpress

Global MEF2 target gene analysis in cardiac and skeletal muscle reveals novel regulation of DUSP6 by p38MAPKMEF2 signaling [ChIP-seq]

Project description:Identfification of MEF2A target genes using ChIP-exo in skeletla muscle and primary cardiomyocytes. Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells. MEF2A target genes were identified in 48 hr DM C2C12 myoblasts cells and primary cardiomyocytes using ChIP-exo. Binding profiles on MEF2A in each cell type were compared. Cross sectional-analysis between ChIP-exo identified targets and RNA-seq analysis of MEF2A deplted myoblasts was also done.

2014-09-09 | E-GEOD-61204 | biostudies-arrayexpress

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