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: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: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
Project description:This experiment employed CUT&Tag-seq (Cleavage Under Targets and Tagmentation with sequencing) to explore the mechanism of how different concentrations of VFAs regulate ruminal epithelial histone modifications under the Grain-diet and Hay-diet patterns in both am and pm. Cells from Grain-am, Grain-pm, Hay-am, and Hay-pm treatment groups were havest for CUT&Tag-seq experiments, n=3 pooled biological replicates per library. The primary histones used for CUT&Tag were Acetyl-Histone H3 (Lys27) Rabbit mAb (H3K27ac, 8173S, CST), Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb (H3K9ac, 9649S, CST), and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (H3K4me3, 9751S, CST).
Project description:To reveal the role of MCM8 in suppressing R-loop accumulation, we performed the CUT&TAG assay using the S9.6 antibody to map genome-wide R-loops in Mcm8 wildtype MEFs and Mcm8 knockout MEFs. We also conducted the CUT&TAG assay to detect genome-wide R-loops in Ddx5 downregulated MEFs by adenovirus infection and in control MEFs. To investigate the underlying molecular mechanism of MCM8 suppressing R-loops, we conducted the DNA sequencing of libraries from CUT&TAG assay using the antibody against FLAG in HEK293 cells transfected with FLAG-MCM8 plasmid and using the S9.6 antibody in HEK293 cells. Besides, an IgG control and control of RNH1 overexpression were included.
Project description:To study the mechanism of cisplatin resistance in uveal melanoma, we performed H3K9ac and H3K27ac Cut&Tag in 92.1-parental and 92.1-resistant uveal melanoma cells.