Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.
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ABSTRACT:
SUBMITTER: Li Y
PROVIDER: S-EPMC8384913 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description: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.
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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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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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Project description:Determining the genomic location of DNA-binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein-DNA interactions at high resolution. In CUT&RUN, a recombinant protein A-microccocal nuclease (pA-MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde-crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA-sequencing libraries that can be sequenced in pools of at least 30. Therefore, CUT&RUN provides an alternative to ChIP-seq approaches for mapping chromatin proteins, which typically have relatively high signal-to-noise ratios, while using fewer cells and at a lower cost. Here, we describe the methods for performing CUT&RUN, generating DNA-sequencing libraries, and analyzing the resulting datasets. © 2019 by John Wiley & Sons, Inc.
| S-EPMC6422702 | biostudies-literature
Project description:Genome-wide chromatin mapping approaches typically focus on one protein at a time. We recently developed multi-CUT&Tag, which enables simultaneous mapping of multiple chromatin proteins in the same single cells or pools of cells. Using barcoded adapters loaded onto antibody-protein A-Tn5 transposase complexes, multi-CUT&Tag marks the locations of each chromatin protein and directly detects colocalization of different proteins in the same cell(s). Although slightly more laborious than CUT&Tag, multi-CUT&Tag provides a powerful option for generating multi-factor maps for epigenomic profiling. For complete details on the use and execution of this protocol, please refer to Gopalan et al. (2021).
| S-EPMC8783141 | biostudies-literature
Project description:Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.
| S-EPMC6598765 | biostudies-literature
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.
| S-EPMC8072009 | biostudies-literature
Project description:Probing epigenomic marks such as histone modifications at a single cell level in thousands of cells has been recently enabled by technologies such as scCUT&Tag. Here we developed a multimodal and optimized iteration of scCUT&Tag called nano-CT (for nano-CUT&Tag) that allows simultaneous probing of three epigenomic modalities at single-cell resolution, using nanobody-Tn5 fusion proteins. nano-CT is compatible with starting materials as low as 25 000 cells and has significantly higher resolution than scCUT&Tag, with a 16-fold increase in the number of fragments per cells. We used nano-CT to simultaneously profile chromatin accessibility, H3K27ac and H3K27me3 in a complex tissue - juvenile mouse brain. The obtained multimodal dataset allowed for discrimination of more cell types/states that scCUT&Tag, and inference of chromatin velocity between ATAC and H3K27ac in the oligodendrocyte (OL) lineage. In addition, we used nano-CT to deconvolute H3K27me3 repressive states and infer two sequential waves of H3K27me3 repression at distinct gene modules during OL lineage progression. Thus, given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in different biological systems at single cell level.
2022-09-20 | GSE198467 | GEO
Project description:Determining chromatin-associated protein localization across the genome has provided insight into the functions of DNA-binding proteins and their connections to disease. However, established protocols requiring large quantities of cell or tissue samples currently limit applications for clinical and biomedical research in this field. Furthermore, most technologies have been optimized to assess abundant histone protein localization, prohibiting the investigation of nonhistone protein localization in low cell numbers. We recently described a protocol to profile chromatin-associated protein localization in as low as one cell: ultra-low-input cleavage under targets and release using nuclease (uliCUT&RUN). Optimized from chromatin immunocleavage and CUT&RUN, uliCUT&RUN is a tethered enzyme-based protocol that utilizes a combination of recombinant protein, antibody recognition and stringent purification to selectively target proteins of interest and isolate the associated DNA. Performed in native conditions, uliCUT&RUN profiles protein localization to chromatin with low input and high precision. Compared with other profiling technologies, uliCUT&RUN can determine nonhistone protein chromatin occupancies in low cell numbers, permitting the investigation into the molecular functions of a range of DNA-binding proteins within rare samples. From sample preparation to sequencing library submission, the uliCUT&RUN protocol takes <2 d to perform, with the accompanying data analysis timeline dependent on experience level.
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