Project description:We developed scNanoSeq-CUT&Tag, a streamlined method by adapting a modified CUT&Tag protocol to Oxford Nanopore sequencing platform for efficient chromatin modification profiling at single-cell resolution. We firstly tested the performance of scNanoSeq-CUT&Tag on six human cell lines: K562, 293T, GM12878, HG002, H9, HFF1 and adult mouse blood cells, it showed that scNanoSeq-CUT&Tag can accurately distinguish different cell types in vitro and in vivo. Moreover, scNanoSeq-CUT&Tag enables to effectively map the allele-specific epigenomic modifications in the human genome andallows to analyze co-occupancy of histone modifications. Taking advantage of long-read sequencing,scNanoSeq-CUT&Tag can sensitively detect epigenomic state of repetitive elements. In addition, by applying scNanoSeq-CUT&Tag to testicular cells of adult mouse B6D2F1, we demonstrated that scNanoSeq-CUT&Tag maps dynamic epigenetic state changes during mouse spermatogenesis. Finally, we exploited the epigenetic changes of human leukemia cell line K562 during DNA demethylation, it showed that NanoSeq-CUT&Tag can capture H3K27ac signals changes along DNA demethylation. Overall, we prove that scNanoSeq-CUT&Tag is a valuable tool for efficiently probing chromatin state changes within individual cells.
Project description:In order to determine that CUT&Tag is similar to known DUX ChIP-seq, we performed CUT&Tag with a mCherry-tagged DUX (with the mCherry antibody). Once confirmed, we pewrformed CUT&Tag for other DUX derivatives with their mCherry tag Then, we performed CUT&Tag for H3K9ac, which is known to globally increase in 2-cell-like cells, which occurs after DUX expression, and CUT&Tag for SMARCC1, a subunit of the SWI/SNF complex
Project description:To investigate how Tbx3 regulates the fate determination of arcuate piptidergic neruons, we performed scRNA-seq, snRNA-seq and CUT&Tag to reveal the function of Tbx3 in fate specification and maintenance of neurons
Project description:In order to explore the molecular mechanism of ZNF200 in the development of NSCLC, we conducted CUT&Tag experiments in the H1299 cell line using IgG and ZNF200 antibodies. Based on previous exploratory experiments, we found an interaction between DDX17 and ZNF200. Therefore, we stably transfected DDX17-HA into H1299 cells and performed CUT&Tag experiments using IgG and ZNF200 antibodies. We then discovered that both ZNF200 and DDX17 act on the promoter region of RBP-J. Consequently, in the DDX17-HA stable transfectants of H1299,H520 and A549 cells, we knocked down ZNF200 and performed CUT&Tag experiments using HA antibodies. At the same time, we knocked down ZNF200 and DDX17 in the H520and A549 cell lines, respectively. Since H3K4me3, as an epigenetic modification, is a hallmark of the active status of gene promoter regions and is usually associated with the activation of gene expression, we subsequently used antibodies against histone H3K4me3 for the CUT&Tag experiments.
Project description:Cleavage Under Targets & Tagmentation (CUT&Tag) is a versatile method for measuring genomic occupancy of chromatin-associated proteins with high sensitivity and specificity. CUT&Tag has low sequencing requirements and is therefore suitable for highly multiplexed experiments, but methods to process samples at throughput without specialized equipment are lacking. Here we present a method for simultaneous parallel processing of 96 CUT&Tag samples in a standard microplate. Plate-CUT&Tag can be carried out in a similar time frame to benchtop CUT&Tag and yields data of comparable quality. We present data from cell culture and patient leukemia samples processed with Plate-CUT&Tag to illustrate its utility in large-scale preclinical and translational studies.
Project description:Precise profiling of epigenomes, including histone modifications and transcription factor binding sites, is essential for better understanding gene regulatory mechanisms. Cleavage Under Targets & Tagmentation (CUT&Tag) is an easy and low-cost epigenomic profiling method that can be performed on a low number of cells and at the single-cell level. A large number of CUT&Tag datasets have been generated in various biological systems, providing a valuable resource. CUT&Tag experiments use the hyperactive transposase Tn5 for tagmentation. We found that the preference of Tn5 captured reads toward accessible chromatin regions can influence the distribution of CUT&Tag reads and cause open chromatin biases, further confounding the analysis of CUT&Tag data. The high sparsity of single-cell sequencing data makes the open chromatin biases more substantial than in bulk sequencing data. Here, we present a comprehensive computational method, PATTY (Propensity Analyzer for Tn5 Transposase Yielded bias), to mitigate the open chromatin bias inherent in CUT&Tag data at both bulk and single-cell levels. By integrating existing transcriptome and epigenome data using machine learning and comprehensive modeling, we demonstrate that PATTY yields more accurate and robust detection of occupancy sites for both active and repressive histone marks than existing methods, with experimental validation. We further designed a single-cell CUT&Tag analysis framework by utilizing this model and showing improved cell clustering from bias-corrected single-cell CUT&Tag data compared to using raw data. This model paved the way for further development of computational tools for improving bulk and single-cell CUT&Tag data analysis.
Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis
Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis