KAS-CUT&Tag for direct mapping of transcription bubbles
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE317877 | GEO | 2026/07/21
REPOSITORIES: GEO
Dataset's files
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| GSE317877_RAW.tar | Raw | |||
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Project description:Transcription is a highly dynamic process that generates the majority of single-stranded DNA in the genome as ‘transcription bubbles’. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq), which marks single-stranded DNA in situ for genome-wide capture and sequencing, based on the fast and specific reaction between N3-kethoxal and guanines in single-stranded DNA in live cells. KAS-seq enables rapid (within 5 min) and sensitive detection of the position, relative level, and dynamics of ssDNA, as a readout of transcriptionally engaged RNA polymerases, with 1,000 cells. KAS-seq defines a group of enhancers that are single-stranded, which confers higher transcription factor binding density, more enhancer-promotor interactions, and higher ability to activate transcription. Under protein condensation inhibition condition, KAS-seq reveals unprecedented, rapid release of RNA polymerase II (Pol II) from a group of promotors, and reveals gene sets with different extents of responses, which correlate with Pol II C-terminal domain (CTD) serine 5 phosphorylation (S5P) level at TSS under no treatment condition. KAS-seq thus facilitates fast, comprehensive, and accurate analysis of transcription dynamics and enhancer activities simultaneously in a low input and high-throughput manner.
2020-02-15 | GSE139420 | GEO
Project description:Kethoxal-assisted ssDNA sequencing (KAS-seq) is gaining popularity as a robust and effective approach to study the dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source, and specific bioinformatic analyzer for KAS-seq data are still lacking. Here we present KAS-Analyzer as a flexible and integrated toolkit to facilitate the analysis and interpretation of KAS-seq data. KAS-Analyzer can perform standard analyses such as quality control, read alignment, and differential RNA polymerase activity analysis. In addition, KAS-Analyzer introduces many novel features, including, but not limited to: calculation of transcriptional indexes, identification of single-stranded transcribing enhancers, and high-resolution mapping of R-loops. We use benchmark datasets to demonstrate that KAS-Analyzer provides a powerful framework to study transient transcriptional regulatory programs. KAS-Analyzer is available at https://github.com/Ruitulyu/KAS-Analyzer.
2022-07-27 | GSE202044 | GEO
Project description:In fully grown oocytes, the genome is considered to be globally transcriptionally silenced. However, this conclusion is primarily derived from the results obtained through immunofluorescence staining or inferred from the highly condensed state of chromosomes, lacking more direct evidence. Here, by using a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, we investigated the landscape of single-strand DNA (ssDNA) throughout the genome and provided a readout of the activity and dynamics of transcription during oocyte meiotic maturation. In non-surrounded nucleolus (NSN) oocytes, we observed a robust KAS-seq signal, indicating the high transcriptional activity. In surrounded nucleolus (SN) oocytes, the presence of ssDNA still persists although the KAS-seq signal was relatively weak, suggesting the presence of transcription. Accompanying with the meiotic resumption, the transcriptional activity gradually decreased, and global repression was detected in matured oocytes. Moreover, we preformed the integrative genomics analysis to dissect the transcriptional dynamics during mouse oocyte maturation. In sum, the present study delineates the detailed transcriptional activity during mammalian oocyte maturation.
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Project description:Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation network and dynamics during cellular processes. In this study, we developed a new KAS-seq (Opti-KAS-seq) procedure, with enhanced efficiency of capturing single-stranded DNA (ssDNA), broader genomic coverage, and adaptability to various sample types. By integrating the highly sensitive Opti-KAS-seq with ATAC-seq, we further introduce KAS-ATAC-seq, a new method that provides quantitative insights into transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks. This feature is particularly adept at identifying ssDNA promoter and Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNA transcripts and specific transcription factors (TFs) binding sites that determine cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes; KAS-ATAC-seq signals exhibit more robust correlation with enhancer activities when compared with ATAC-seq data and active histone mark profiles. Our analysis of promoters and SSTEs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to RA treatment. We further discovered that ETS TFs and YY1 are critical in initiating early neural differentiation from mESCs to NPCs. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes and biomedical applications.
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Project description:Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation network and dynamics during cellular processes. In this study, we developed a new KAS-seq (Opti-KAS-seq) procedure, with enhanced efficiency of capturing single-stranded DNA (ssDNA), broader genomic coverage, and adaptability to various sample types. By integrating the highly sensitive Opti-KAS-seq with ATAC-seq, we further introduce KAS-ATAC-seq, a new method that provides quantitative insights into transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks. This feature is particularly adept at identifying ssDNA promoter and Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNA transcripts and specific transcription factors (TFs) binding sites that determine cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes; KAS-ATAC-seq signals exhibit more robust correlation with enhancer activities when compared with ATAC-seq data and active histone mark profiles. Our analysis of promoters and SSTEs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to RA treatment. We further discovered that ETS TFs and YY1 are critical in initiating early neural differentiation from mESCs to NPCs. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes and biomedical applications.
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Project description:Gene regulation in most eukaryotes involves two fundamental physical processes -- alterations in the packaging of the genome by nucleosomes, with active cis-regulatory elements (CREs) generally characterized by an open-chromatin configuration, and the activation of transcription. Mapping these physical properties and biochemical activities genome-wide -- through profiling chromatin accessibility and active transcription -- have now long been key tools used to understand the logic and mechanisms of transcription and its regulation. However, the direct relationship between the two has not been accessible to measurements, leaving the exact chromatin state of actively transcribed DNA molecules unresolved. To address this question, we developed KAS-ATAC, a combination of the KAS-seq (Kethoxal-Assisted SsDNA sequencing and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) methods for mapping single-stranded DNA (and thus active transcription) and chromatin accessibility, respectively, which maps DNA fragments that are simultaneously accessible and containing ssDNA genome-wide. We use KAS-ATAC to evaluate levels of active transcription over different classes of regulatory elements in the human genome, to estimate the absolute levels of transcribed accessible DNA over CREs, to map the nucleosomal configurations associated with RNA polymerase activities, and to assess transcription factor association with transcribed DNA through transcription factor binding site (TFBS) footprinting.
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