RELACS nuclei barcoding enables high-throughput ChIP-seq

Arrigoni, Laura; Al-Hasani, Hoor; Ramírez, Fidel; Panzeri, Ilaria; Ryan, Devon P.; Santacruz, Diana; Kress, Nadia; Pospisilik, J. Andrew; Bönisch, Ulrike

doi:10.1038/s42003-018-0219-z

Cited by 31 publications

(55 citation statements)

References 46 publications

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“…Barcoding-first ChIP techniques have gained popularity because of the combined benefit of multiplexing and quantitative readout [11][12][13][14] . Pooling barcoded samples greatly increases throughput without complicated automation, while effectively removing technical variability between samples.…”

Section: Resultsmentioning

confidence: 99%

Quantitative multiplexed ChIP reveals global alterations that shape promoter bivalency in ground state embryonic stem cells

Kumar

ElsaÌˆsser

2019

Preprint

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To understand the epigenetic foundation of naïve pluripotent cells, we implement a quantitative multiplexed ChIP-Seq method (MINUTE-ChIP) comparing mouse ESC grown in 2i versus Serum conditions. Combined with quantitative western blot and mass spectrometry, we find compelling evidence for a broad H3K27me3 hypermethylation of the genome, concomitant with the widespread loss of DNA CpG methylation. We show that opposing action of EZH2 and JMJD3/UTX shape the H3K27me3 landscape, with almost all bivalent promoters stably retaining high H3K27me3 levels in 2i. In parallel, we show a mechanistically uncoupled, global decrease of H3K4me3 and strong reduction at bivalent promoters, suggesting that low H3K4me3 and high H3K27me3 levels at bivalent promoters safeguard naïve pluripotency.

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Section: Resultsmentioning

confidence: 99%

Quantitative multiplexed ChIP reveals global alterations that shape promoter bivalency in ground state embryonic stem cells

Kumar

ElsaÌˆsser

2019

Preprint

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“…Future efforts to further reduce the extent of this degradation likely will increase the ChIP signal even more. For example, as an alternative to sonication, the DNA can also be fragmented enzymatically using DNases such as MNase (Skene & Henikoff, 2015;Arrigoni et al, 2018;Gutin et al, 2018). This approach could prevent the degradation that was observed during sonication and may subsequently increase the ChIP signal.…”

Section: Strategies That Reduce Protein Degradation Increase Chip Signalmentioning

confidence: 99%

“…For example, optimizations that additionally reduce the number of steps or pool the samples early in the protocol could be beneficial to further reduce the variation between replicates. An example of such a strategy is barcoding of the DNA while it is bound to the beads (Lara-Astiaso et al, 2014;Schmidl et al, 2015;Wallerman et al, 2015;Gutin et al, 2018) or fragmentation and barcoding of DNA while still in the nucleus (Arrigoni et al, 2018). Alternatively, many orthogonal method exist that quantify protein-DNA binding, which may be used if no clear ChIP signal can be obtained (Galas & Schmitz, 1978;Garner & Revzin, 1981;Smith, 1985;Nick & Gilbert, 1985;Ellington & Szostak, 1990;Tuerk & Gold, 1990;Wang & Reed, 1993;Yin et al, 1995;van Steensel & Henikoff, 2000;Bulyk et al, 2001;Schmid et al, 2004;Cremazy et al, 2005;Lushnikov et al, 2006;Maerkl & Quake, 2007;Zentner et al, 2015;Skene & Henikoff, 2017).…”

Section: Limitations and Outlookmentioning

confidence: 99%

An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

Jonge

Brok

Kemmeren

et al. 2019

Preprint

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Chromatin immunoprecipitation (ChIP) is a commonly used technique to investigate which parts of a genome are bound by a particular protein. The result of ChIP is often interpreted in a binary manner: bound or not bound. Due to this focus, ChIP protocols frequently lack the ability to quantitatively compare samples with each other, for example in a time series or under different growth conditions. Here, using the yeast S. cerevisiae transcription factors Cbf1, Abf1, Reb1, Mcm1 and Sum1, we optimized the five major steps of a commonly used ChIP protocol: cross-linking, quenching, cell lysis, fragmentation and immunoprecipitation. Quenching with glycine is inefficient and can lead to large degrees of variability, an issue that is resolved by using tris(hydroxymethyl)aminomethane (Tris). Another source of variability is degradation of the protein of interest during the procedure. Enzymatic cell lysis with zymolyase can lead to extensive protein degradation, which is greatly reduced by mechanical lysis through bead beating. Degradation also occurs during sonication of chromatin, affecting large proteins in particular. An optimal mix of protease inhibitors and cross-linking with a higher percentage of formaldehyde reduces the extent of this degradation. Finally we also show that the immunoprecipitation step itself can be greatly improved with magnetic beads and optimized incubation/washing steps. The study results in a highly optimized protocol, which is shorter, easier to perform and has a stronger, more reproducible signal with less background. This protocol is presented in detail. In addition, the results highlight the greatest sources of variability in many other protocols, showing which steps are important to focus on for reproducible and quantitatively comparable ChIP experiments.

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“…Over the last ten years, much work has been devoted to address these and other shortcomings (4–8). In line with these efforts, we have recently developed RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ ), a method that employs chromatin barcoding to enable high-throughput generation of ChIP-seq experiments (9). RELACS works reliably with low input material and can be used for quantitative ChIP-seq analysis (9,10).…”

Section: Introductionmentioning

confidence: 99%

AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq

Arrigoni

Ferrari

Weller

et al. 2020

Preprint

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11Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method used to profile 12 protein-DNA interactions genome-wide. RELACS (Restriction Enzyme-based Labeling of 13 Chromatin in Situ) is a recently developed ChIP-seq protocol that deploys a chromatin barcoding 14 strategy to enable standardized and high-throughput generation of ChIP-seq data. The manual 15 implementation of RELACS is constrained by human processivity in both data generation and data 16 analysis. To overcome these limitations, we have developed AutoRELACS, an automated 17 implementation of the RELACS protocol using the liquid handler Biomek i7 workstation. We 18 match the unprecedented processivity in data generation allowed by AutoRELACS with the 19 automated computation pipelines offered by snakePipes. In doing so, we build a continuous 20 workflow that streamlines epigenetic profiling, from sample collection to biological interpretation. 21Here, we show that AutoRELACS successfully automates chromatin barcode integration, and is 22 able to generate high-quality ChIP-seq data comparable with the standards of the manual protocol, 23 also for limited amounts of biological samples. 24 25 2 BACKGROUND 26Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used method to 27 study protein-DNA interactions genome-wide (1). Despite the enormous contribution that ChIP-28 seq has brought to our understanding of epigenetic and transcriptional control, the traditional ChIP-29 seq protocol (2,3) presents various limitations. For example, it requires substantial amounts of 30 biological input material, which is often a limiting factor in relevant clinical settings, and it is low-31 throughput, which prevents comprehensive epigenetic profiling. Furthermore, the protocol is 32 poorly standardized across cell types, resulting in a high degree of technical variability that 33 hampers biological interpretation of the data. 35Over the last ten years, much work has been devoted to address these and other shortcomings (4-36 8). In line with these efforts, we have recently developed RELACS (Restriction Enzyme-based 37 Labeling of Chromatin in Situ), a method that employs chromatin barcoding to enable high-38 throughput generation of ChIP-seq experiments (9). RELACS works reliably with low input 39 material and can be used for quantitative ChIP-seq analysis (9,10). The method is highly 40 standardized, and could potentially be scaled to profile hundreds of samples in parallel for tens of 41 DNA-binding proteins at once. Yet, the current manual implementation is limited by human 42 processivity in both data generation and data analysis. 43 44To match the ideal potential of this methodology, we have implemented an automated version of 45 the RELACS protocol, named AutoRELACS, using the liquid handler Biomek i7 automated 46 workstation (Beckman Coulter). While other automated ChIP-seq implementations already exist 47 (11,12), they still require a large amount of sample material, and they do not utilize the enormous 48 3 multiple...

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RELACS nuclei barcoding enables high-throughput ChIP-seq

Cited by 31 publications

References 46 publications

Quantitative multiplexed ChIP reveals global alterations that shape promoter bivalency in ground state embryonic stem cells

Quantitative multiplexed ChIP reveals global alterations that shape promoter bivalency in ground state embryonic stem cells

An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq

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