Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Berguet, Geoffrey; Hendrickx, Jan; Sabatel, Céline; Laczik, Miklos; Squazzo, Sharon L.; Peláez, Ignacio Mazón; Saxena, Rini; Pendeville, Hélène; Poncelet, Dominique

doi:10.3791/52150

Cited by 5 publications

(2 citation statements)

References 11 publications

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“…Addressing the hierarchy and multifactor issues of cofactor squelching would require a dedicated and systematic high throughput approach for screening of multiple combinations of cofactors for the ability to rescue signal‐repressed gene expression upon overexpression. While this is a challenging, task the recent advances in CRISPR/cas9 technologies has enabled the simultaneous activation of multiple endogenous genes with a defined stoichiometry , and strategies for high throughput, multiplexed analysis of cofactor binding and transcriptional activity have also emerged .…”

Section: Unresolved Questionsmentioning

confidence: 99%

Cofactor squelching: Artifact or fact?

et al. 2016

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Cofactor squelching is the term used to describe competition between transcription factors (TFs) for a limited amount of cofactors in a cell with the functional consequence that TFs in a given cell interfere with the activity of each other. Since cofactor squelching was proposed based primarily on reporter assays some 30 years ago, it has remained controversial, and the idea that it could be a physiologically relevant mechanism for transcriptional repression has not received much support. However, recent genome-wide studies have demonstrated that signal-dependent TFs are very often absent from the enhancers that are acutely repressed by those signals, which is consistent with an indirect mechanism of repression such as squelching. Here we review these recent studies in the light of the classical studies of cofactor squelching, and we discuss how TF cooperativity in so-called hotspots and super-enhancers may sensitize these to cofactor squelching.

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Section: Unresolved Questionsmentioning

confidence: 99%

Cofactor squelching: Artifact or fact?

et al. 2016

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“…Each of the various ChIP-seq methodologies yield incremental benefits, but suffer from (a combination of) low read complexity, lack of robustness, suboptimal throughput and lengthy and/or laborious protocols. On the other hand, semi-automated workflows have been developed to increase reproducibility of ChIP-Seq and reduce the workload of the laborious protocol (Aldridge et al 2013;Berguet et al 2014;Gasper et al 2014;Wallerman et al 2015), but these generally require high quantities of input material. Recent studies have shown the feasibility of combining low input samples with automated workflows using microfluidic devices (Cao et al 2015;Shen et al 2015;Murphy et al 2018).…”

Section: Introductionmentioning

confidence: 99%

A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation

Dirks

Thomas

Jones

et al. 2020

Preprint

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Epigenetic profiling by ChIP-Seq has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-Seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput.Although prototypic microfluidic ChIP-Seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-Seq profiling of low-input samples, we constructed PDMS-based plates containing microfluidic Integrated Fluidic Circuits capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time. These disposable plates can conveniently be loaded into a widely available controller for pneumatics and thermocycling, making the ChIP-Seq procedure Plug and Play (PnP). We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for multiple widely-profiled post-translational histone modifications, together allowing genome-wide identification of regulatory elements. As proof of principle, we managed to generate high-quality epigenetic profiles of rare totipotent subpopulations of mESCs using our platform. In light of the ready-to-go ChIP plates and the automated workflow, we named our procedure PnP-ChIP-Seq.PnP-ChIP-Seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-Seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.

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A plug and play microfluidic platform for standardized sensitive low-input chromatin immunoprecipitation

Dirks

Thomas

et al. 2021

Genome Res.

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Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time and 4.5 hours machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the Plug and Play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naïve and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the 2-cell stage of embryonic development. PnP-ChIP-seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE120673.

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Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Cited by 5 publications

References 11 publications

Cofactor squelching: Artifact or fact?

Cofactor squelching: Artifact or fact?

A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation

A plug and play microfluidic platform for standardized sensitive low-input chromatin immunoprecipitation

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