Mapping Histone Modifications in Low Cell Number and Single Cells Using Antibody-guided Chromatin Tagmentation (ACT-seq) (original) (raw)
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eLife, 2021
Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal s...
Antibody-guided Chromatin Tagmentation for Two or More Factors (ACT2-seq)
2021
This protocol details the reagents and steps required to perform antibody-guided chromatin tagmentation for two or more factors (ACT2-seq, ACT2). Like its predecessor ACT-seq, ACT2 uses a fusion of protein A and Tn5 transposase to bind and profile epigenetic marks across the genome. ACT2 builds on the capabilities of ACT-seq by directly and concurrently profiling co-occupancy of epigenetic marks, which previously required laborious, expensive, and technically challenging approaches involving fluorescence, magnetic beads, or single-cell methods. ACT2 requires only standard pipetting and centrifugation techniques and can be completed in less than a single day of bench work.
TAF-ChIP: an ultra-low input approach for genome-wide chromatin immunoprecipitation assay
Life Science Alliance
Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) is a powerful technique to study transcriptional regulation. However, the requirement of millions of cells to generate results with high signal-to-noise ratio precludes it in the study of small cell populations. Here, we present a tagmentation-assisted fragmentation ChIP (TAF-ChIP) and sequencing method to generate high-quality histone profiles from low cell numbers. The data obtained from the TAF-ChIP approach are amenable to standard tools for ChIP-Seq analysis, owing to its high signal-to-noise ratio. The epigenetic profiles from TAF-ChIP approach showed high agreement with conventional ChIP-Seq datasets, thereby underlining the utility of this approach.
Science (New York, N.Y.), 2015
Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas.
F1000 - Post-publication peer review of the biomedical literature, 2018
Post-translational modifications of histone proteins and exchanges of histone variants at chromatin are central to the regulation of nearly all DNA-templated biological processes. However, the degree and variability of chromatin modifications in specific human immune cells remain largely unknown. Here we employ a highly multiplexed mass cytometry analysis to profile the global levels of a broad array of chromatin modifications in primary human immune cells at the singlecell level. Our data reveal markedly different cell type-and hematopoietic lineage-specific
Proceedings of the National Academy of Sciences of the United States of America, 2010
Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed, paraffin-embedded material. Here, we present a methodology [pathology tissue-ChIP (PAT-ChIP)] to extract and immunoprecipitate chromatin from paraffin-embedded patient samples up to several years old. In a pairwise comparison with canonical ChIP, PAT-ChIP showed a high reproducibility of results for several histone marks and an identical ability to detect dynamic changes in chromatin structure upon pharmacological treatment. Finally, we showed that PAT-ChIP can be coupled with high-throughput sequencing (PAT-ChIP-Seq) for the genome-wide analysis of distinct chromatin modifications. PAT-ChIP therefore represents a versatile procedure and diagnostic tool for the analysis of epigenetic alterations in c...
2022
SummaryJoint profiling of chromatin accessibility and gene expression from the same single cell/nucleus provides critical information about cell types in a tissue and cell states during a dynamic process. These emerging multi-omics techniques help the investigation of cell-type resolved gene regulatory mechanisms1–7. However, many methods are currently limited by low sensitivity, low throughput or complex workflow. Here, we developed in situ SHERRY after ATAC-seq (ISSAAC-seq), a highly sensitive and flexible single cell multi-omics method to interrogate chromatin accessibility and gene expression from the same single nucleus. We demonstrated that ISSAAC-seq is sensitive and provides high quality data with orders of magnitude more features than existing methods. Using the joint profiles from over 10,000 nuclei from the mouse cerebral cortex, we uncovered major and rare cell types and cell-type specific regulatory elements and identified heterogeneity at the chromatin level within est...
Iterative Fragmentation Improves the Detection of ChIP-seq Peaks for Inactive Histone Marks
Bioinformatics and Biology Insights, 2016
As chromatin immunoprecipitation (ChIP) sequencing is becoming the dominant technique for studying chromatin modifications, new protocols surface to improve the method. Bioinformatics is also essential to analyze and understand the results, and precise analysis helps us to identify the effects of protocol optimizations. We applied iterative sonication-sending the fragmented DNA after ChIP through additional round(s) of shearing-to a number of samples, testing the effects on different histone marks, aiming to uncover potential benefits of inactive histone marks specifically. We developed an analysis pipeline that utilizes our unique, enrichment-type specific approach to peak calling. With the help of this pipeline, we managed to accurately describe the advantages and disadvantages of the iterative refragmentation technique, and we successfully identified possible fields for its applications, where it enhances the results greatly. In addition to the resonication protocol description, we provide guidelines for peak calling optimization and a freely implementable pipeline for data analysis.
International Journal of Molecular Sciences
Epigenetic regulators play a crucial role in establishing and maintaining gene expression states. To date, the main efforts to study cellular heterogeneity have focused on elucidating the variable nature of the chromatin landscape. Specific chromatin organisation is fundamental for normal organogenesis and developmental homeostasis and can be affected by different environmental factors. The latter can lead to detrimental alterations in gene transcription, as well as pathological conditions such as cancer. Epigenetic marks regulate the transcriptional output of cells. Centromeres are chromosome structures that are epigenetically regulated and are crucial for accurate segregation. The advent of single-cell epigenetic profiling has provided finer analytical resolution, exposing the intrinsic peculiarities of different cells within an apparently homogenous population. In this review, we discuss recent advances in methodologies applied to epigenetics, such as CUT&RUN and CUT&TAG. Then, w...