Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ

  title={Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ},
  author={Tong Wu and Ruitu Lyu and Qiancheng You and Chuan He},
  journal={Nature methods},
  pages={515 - 523}
Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as ‘transcription bubbles’. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N 3 -kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells… 

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The recent study shows that ssDNA signals measured by KAS-seq simultaneously reveal the dynamics of transcriptionally engaged RNA polymerase (Pol) II, transcribing enhancers, RNA Pol I and Pol III activities and potentially non-canonical DNA structures with high analytical sensitivity.

KAS-pipe2: a flexible toolkit for exploring KAS-seq and spKAS-seq data

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Ligand-induced native G-quadruplex stabilization impairs transcription initiation

A reciprocal genome-wide regulation between native G4 dynamics and gene transcription is revealed, which will deepen the understanding of G4 biology toward therapeutically targeting G4s in human diseases.

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BisMapR: a strand-specific, nuclease-based method for genome-wide R-loop detection

BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression, and achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies.

Chromophore-assisted proximity labeling of DNA reveals chromosomal organization in living cells.

A novel proximity-dependent DNA labeling method based on the chromophore-assisted nucleobase photooxidation that achieves blue light-activated labeling of local DNA with a bioorthogonal functional handle and is generally applicable for the genome-wide high-resolution mapping of the spatial chromosome organization in living cells.

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Selection and thermostability suggest G-quadruplexes are novel functional elements of the human genome

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Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) for capturing transcription dynamics and enhancer activity

A rapid and sensitive labeling of single-stranded DNA for sequencing based on a kethoxal-guanine reaction that enables rapid measurement of the dynamics of transcriptionally-engaged Pol II, transcribing enhancers, Pol I and Pol III activities, and non-canonical DNA structures involving ssDNA simultaneously in situ.

Nascent transcript sequencing visualizes transcription at nucleotide resolution

An approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution is presented, revealing pervasive polymerase pausing and backtracking throughout the body of transcripts.

Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing

A precision nuclear run-on and sequencing assay to map the genome-wide distribution of transcriptionally engaged Pol II at base pair resolution and shows how the promoter dictates transcriptional pausing and detects the preferential localization of active transcription complexes within the genome.

Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters

Global run-on sequencing, GRO-seq, shows that peaks of promoter-proximal polymerase reside on ∼30% of human genes, transcription extends beyond pre-messenger RNA 3′ cleavage, and antisense transcription is prevalent.

Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

The feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making is demonstrated and classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes are discovered.

Mapping of RNA polymerase on mammalian genes in cells and nuclei.

Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei are explored for the first time in vivo.

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

A novel method is developed—single-stranded DNA (ssDNA) sequencing (SSDS)—that specifically detects protein-bound single-strander DNA at DSB ends that reduces the nonspecific double-stranding DNA (dsDNA) background >10-fold.