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Efficient low-cost chromatin profiling with CUT&Tag.

ABSTRACT: We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters ('tagmentation') for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.

SUBMITTER: Kaya-Okur HS

PROVIDER: S-EPMC8318778 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Efficient chromatin profiling of H3K4me3 modification in cotton using CUT&Tag.

Project description:BackgroundIn 2019, Kaya-Okur et al. reported on the cleavage under targets and tagmentation (CUT&Tag) technology for efficient profiling of epigenetically modified DNA fragments. It was used mainly for cultured cell lines and was especially effective for small samples and single cells. This strategy generated high-resolution and low-background-noise chromatin profiling data for epigenomic analysis. CUT&Tag is well suited to be used in plant cells, especially in tissues from which small samples are taken, such as ovules, anthers, and fibers.ResultsHere, we present a CUT&Tag protocol step by step using plant nuclei. In this protocol, we quantified the nuclei that can be used in each CUT&Tag reaction, and compared the efficiency of CUT&Tag with chromatin immunoprecipitation with sequencing (ChIP-seq) in the leaves of cotton. A general workflow for the bioinformatic analysis of CUT&Tag is also provided. Results indicated that, compared with ChIP-seq, the CUT&Tag procedure was faster and showed a higher-resolution, lower-background signal than did ChIP.ConclusionA CUT&Tag protocol has been refined for plant cells using intact nuclei that have been isolated.

| S-EPMC7460760 | biostudies-literature

Low-cost high-resolution chromatin profiling with CUT&Tag

Project description:We recently introduced CUT&Tag, an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei, and then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds DNA sequencing adapters (“tagmentation”) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses exposure artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves signal-to-noise over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube from cells to amplified libraries, providing low-cost high-resolution genome-wide chromatin maps. By simplifying library preparation, CUT&Tag requires less than a day at the bench from live cells to sequencing-ready barcoded libraries. Because of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for ~$25 per sample, enabling routine genome-wide profiling of chromatin proteins and modifications that requires no special skills or equipment.

2020-07-15 | GSE145187 | GEO

Low-cost high-resolution chromatin profiling with CUT&Tag

Project description:Low-cost high-resolution chromatin profiling with CUT&Tag

| PRJNA606273 | ENA

Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia.

Project description:Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.

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Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level.

Project description:Spatial omics emerged as a new frontier of biological and biomedical research. Here, we present spatial-CUT&Tag for spatially resolved genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding, and next-generation sequencing. Spatially resolved chromatin states in mouse embryos revealed tissue-type-specific epigenetic regulations in concordance with ENCODE references and provide spatial information at tissue scale. Spatial-CUT&Tag revealed epigenetic control of the cortical layer development and spatial patterning of cell types determined by histone modification in mouse brain. Single-cell epigenomes can be derived in situ by identifying 20-micrometer pixels containing only one nucleus using immunofluorescence imaging. Spatial chromatin modification profiling in tissue may offer new opportunities to study epigenetic regulation, cell function, and fate decision in normal physiology and pathogenesis.

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CUT&Tag-BS: an efficient and low-cost method for simultaneous profiling of histone modification and DNA methylation

Project description:It remains a challenge to decipher the complex relationship between DNA methylation, histone modification, and the underlying DNA sequence with limited input material. Here, we developed an efficient, low-input, and low-cost method for simultaneous profiling of genomic binding sites of histone modification and methylation status of the underlying DNA at single-base resolution from the same cells in a single experiment by integrating CUT&Tag with tagmentation-based bisulfite sequencing (CUT&Tag-BS). We demonstrated the validity of our method for both active and repressive histone modifications using 250,000 mouse ESCs. CUT&Tag-BS showed similar enrichment patterns of histone modification to those observed in non-bisulfite-treated control; it further revealed that H3K4me1-marked regions are mostly CpG-poor, lack of methylation concordance, and exhibit prevalent DNA methylation heterogeneity among the cells. We anticipate that CUT&Tag-BS will be widely applied to directly address the genomic relationship between DNA methylation and histone modification, especially in low-input scenario with precious biological samples.

2021-10-20 | GSE179266 | GEO

CUT&Tag-BS for simultaneous profiling of histone modification and DNA methylation with high efficiency and low cost.

Project description:It remains a challenge to decipher the complex relationship between DNA methylation, histone modification, and the underlying DNA sequence with limited input material. Here, we developed an efficient, low-input, and low-cost method for the simultaneous profiling of genomic localization of histone modification and methylation status of the underlying DNA at single-base resolution from the same cells in a single experiment by integrating cleavage under targets and tagmentation (CUT&Tag) with tagmentation-based bisulfite sequencing (CUT&Tag-BS). We demonstrated the validity of our method using representative histone modifications of euchromatin and constitutive and facultative heterochromatin (H3K4me1, H3K9me3, and H3K27me3, respectively). Similar histone modification enrichment patterns were observed in CUT&Tag-BS compared with non-bisulfite-treated control, and H3K4me1-marked regions were found to mostly be CpG poor, lack methylation concordance, and exhibit prevalent DNA methylation heterogeneity among mouse embryonic stem cells (mESCs). We anticipate that CUT&Tag-BS will be widely applied to directly address the genomic relationship between DNA methylation and histone modification, especially in low-input scenarios with precious biological samples.

| S-EPMC8754398 | biostudies-literature

CUT&Tag for efficient epigenomic profiling of small samples and single cells.

Project description:Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

| S-EPMC6488672 | biostudies-literature

Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.

Project description:Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34+ hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021).

| S-EPMC8384913 | biostudies-literature

Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag.

Project description:Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

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