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In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 106 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni, whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells with 30,000-300,000 cells as optimum. We also present a streamlined protocol for the preparation of ChIP input libraries.

In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 106 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni, whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells. We also present a streamlined protocol for the preparation of ChIP input libraries.

Abstract Background Chromatin immunoprecipitation coupled to sequencing (ChIP-seq) is widely used to map histone modifications and transcription factor binding on a genome-wide level. Results We present high-throughput ChIPmentation (HT-ChIPmentation) that eliminates the need for DNA purification prior to library amplification and reduces reverse-crosslinking time from hours to minutes. Conclusions The resulting workflow is easily established, extremely rapid, and compatible with requirements for very low numbers of FACS sorted cells, high-throughput applications and single day data generation.

Whole-genome duplications (WGD) have been considered as springboards that potentiate lineage diversification through increasing functional redundancy. Divergence in gene regulatory elements is a central mechanism for evolutionary diversification, yet the patterns and processes governing regulatory divergence following events that lead to massive functional redundancy, such as WGD, remain largely unknown. We studied the patterns of divergence and strength of natural selection on regulatory elements in the Atlantic salmon (Salmo salar) genome, which has undergone WGD 100–80 Ma. Using ChIPmentation, we first show that H3K27ac, a histone modification typical to enhancers and promoters, is associated with genic regions, tissue-specific transcription factor binding motifs, and with gene transcription levels in immature testes. Divergence in transcription between duplicated genes from WGD (ohnologs) correlated with difference in the number of proximal regulatory elements, but not with promoter elements, suggesting that functional divergence between ohnologs after WGD is mainly driven by enhancers. By comparing H3K27ac regions between duplicated genome blocks, we further show that a longer polyploid state post-WGD has constrained regulatory divergence. Patterns of genetic diversity across natural populations inferred from resequencing indicate that recent evolutionary pressures on H3K27ac regions are dominated by largely neutral evolution. In sum, our results suggest that post-WGD functional redundancy in regulatory elements continues to have an impact on the evolution of the salmon genome, promoting largely neutral evolution of regulatory elements despite their association with transcription levels. These results highlight a case where genome-wide regulatory evolution following an ancient WGD is dominated by genetic drift. [ABSTRACT FROM AUTHOR]

In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 10 6 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni , whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells with 30,000-300,000 cells as optimum. We also present a streamlined protocol for the preparation of ChIP input libraries., Competing Interests: Competing interests: Diagenode is the company which commercialized the ChIPmentation technology. ACV and AZS are employees of Diagenode., (Copyright: © 2024 Lasica C et al.)

Background: Chromatin immunoprecipitation coupled to sequencing (ChIP-seq) is widely used to map histone modifications and transcription factor binding on a genome-wide level. Results: We present high-throughput ChIPmentation (HT-ChIPmentation) that eliminates the need for DNA purification prior to library amplification and reduces reverse-crosslinking time from hours to minutes. Conclusions: The resulting workflow is easily established, extremely rapid, and compatible with requirements for very low numbers of FACS sorted cells, high-throughput applications and single day data generation. [ABSTRACT FROM AUTHOR]

Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (>10 7 ) as starting material, which are often impossible to obtain for rare immune populations. Here we describe a streamlined ChIP protocol optimised for small cell numbers in conjunction with transposon-tagging mediated sequencing library preparation (ChIPmentation) which allows the analysis of samples of as low as 10 5 cells.

DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.

Many of the key cellular processes including establishing the cell's identity are governed by chromatin proteins. Mapping their binding on the level of a single cell would give us important insights into a new dimension of cellular heterogeneity. However, ChIP-seq, the main method to study protein-DNA interaction in the chromatin context, has proven very challenging to scale down. ChIPmentation is a modification of ChIP-seq, in which the Tn5 transposase is used to introduce sequencing adapters in one step. This allows to significantly reduce the required input material. ChIPmentation is a robust and versatile approach and even though it has not yet achieved single-cell resolution, we believe that it is a very promising starting point for further downscaling.