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: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:This study aimed to adapt CUT&Tag to Plasmodium falciparum samples as an efficient and sensitive alternative to classical ChIP-sequencing. We compare H3K9me3 and HP1 CUT&Tag with ChIP-seq datasets, showing successful establishment of CUT&Tag in P. falciparum. Next we aimed to scale down required input material for our CUT&Tag reactions and generated high-quality HP1 tracks with as little as 10.000 nuclei. To minimise potential sample loss we tested feasibility of utilising (frozen) saponin parasite isolates as input material instead of nuclei, which proved to be viable. Lastly, we deployed our new technique Dimerisation-induced Biotinylation-CUT&Tag (DiBioCUT&Tag) to catch transient interactions by biotinylation of strongly associated proteins such as histones. We tested this technique on HP1 and compared standart CUT&Tag with DiBioCUT&Tag. Furthermore, we explored interactions of the transcription factor BDP5, which we were previously unable to succesfully ChIP.
Project description:To investigate the genome-wide transcription targets of GR in renal cell carcinoma, cleavage under targets and tagmentation (CUT&Tag) was performed in Caki-1 cells using antibodies againstGR. Following CUT&Tag, DNAs were amplified using non-biased conditions, labeled, and sequenced with Illumina NovaSeq 150PE.
Project description:Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed in situ chromatin profiling strategy that is rapidly replacing precipitation-based methods. The efficiency of the method enabled chromatin profiling in single cells but is limited by the numbers of cells that can be profiled. Here, we describe a combinatorial barcoding strategy for CUT&Tag that harnesses a nanowell dispenser for simple, high-resolution high-throughput single-cell chromatin profiling. We describe a pipeline for single-cell indexed CUT&Tag (sciCUT&Tag) that uses SNPs to facilitate doublet-cell removal and minimize batch effects. We illustrate the optimized protocol by analysis of mouse and human cell lines, as well as human peripheral blood mononuclear cells. We have also used sciCUT&Tag for simultaneous profiling of multiple chromatin epitopes in single cells. The reduced cost, improved resolution and scalability of sciCUT&Tag make it an attractive platform to profile chromatin features in single cells.
Project description:Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and inference of cell type-specific gene expression. In sum, multi-CUT&Tag increases the “per cell” information content of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.