NanoTag - an IgG-free method for mapping DNA-protein interactions
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PROVIDER: PRJEB77284 | ENA |
REPOSITORIES: ENA
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Project description:Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS). 30,000-50,000 GFP positive and negative cells were collected from FACS for ATAC-seq. ATAC-seq libraries were created with Tn5 transposase and single-end sequenced on the Illumina HiSeq 2500 platform
2018-08-21 | E-MTAB-6078 | biostudies-arrayexpress
Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries form little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.
2017-11-13 | GSE101519 | GEO
Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries from little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.
2017-11-13 | GSE101518 | GEO
Project description:Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.
2024-12-15 | GSE283706 | GEO
Project description:Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.
2024-12-15 | GSE283705 | GEO
Project description:Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.
2024-12-15 | GSE283704 | GEO
Project description:Tn5 transposase is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes, but its DNA target site in chromatin has not been understood. In the present study, the well-positioned dinucleosomes were reconstituted, and the Tn5 transposase target sites were mapped in the dinucleosomes in vitro. We found that Tn5 transposase preferentially targets near the entry-exit DNA region within the nucleosome, if the linker DNA exists between two nucleosomes. This specific DNA targeting by Tn5 did not depend on the linker DNA length and DNA sequence. Tn5 transposase becomes to target the middle of the linker DNA, in addition to the entry-exit site of the nucleosome, if the linker DNA length extends to 30 base pairs. These in vitro data provide direct evidence for the Tn5 target sites in the nucleosome, resulting important information for interpretation of the Tn5-transposase-based genomics methods, which have been interpreted as linker or nucleosome-free DNA regions in genomes.
2019-09-18 | GSE130322 | GEO
Project description:Chromatin accessibility captures the binding status of protein factors to chromosomes in vivo, and has been considered a highly informative proxy for functional protein-DNA interactions. Existing DNase I and Tn5 transposase based assays generally require tens of thousands to millions of fresh cells. Applying Tn5 tagmentation to single cells yields very sparse maps. Here we present a transposome hypersensitive sites sequencing assay (THS-seq) for highly sensitive characterizations of chromatin accessibility.
2016-01-09 | GSE72089 | GEO
Project description:A total of 50,000 viable cells were used to resuspend and isolate nuclei; then, chromatin was fragmented using Tn5 transposase and amplified. Last, the library was purified and the concentration was measured.
2022-01-01 | E-MTAB-11023 | biostudies-arrayexpress
Project description:In this experiment we test our recently developed CLIP2C method for the identification of the RNA molecules bound by specific RNA binding proteins. CLIP2C, involves a first round of 2C, the treatment of the eluates with DNase I and an RNA fragmentation step. After this, a small aliquot is separated to be sequenced as an input, while the rest is subjected for immunoprecipitation of the protein of interest. Libraries from the input and the RNA isolated from the immunoprecipitation are generated and sequenced. In this particular experiment we used Pab1-TAP and Tdh3-Protein A tagged strains to be used in IgG-based pull-downs. An untagged WT strain was also included in the experiment as a negative control. Sequencing data from the inputs was used to address the variability in gene expression between strains and sequencing data from the IPs to detect enriched target genes or regions.
2021-05-28 | E-MTAB-9263 | biostudies-arrayexpress