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Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).

ABSTRACT: Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).

PROVIDER: PRJNA807272 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18034815.fastq.gz	Fastqsanger.gz
	SRR18034816_1.fastq.gz	Fastqsanger.gz
	SRR18034816_2.fastq.gz	Fastqsanger.gz

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Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).

Project description:Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).

2022-02-18 | GSE196761 | GEO

DNA molecular combing-based replication fork directionality profiling

Project description:The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas ineffective origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing (OK-seq). Importantly, we demonstrate that inefficient origins do not manifest as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

2021-03-16 | GSE164252 | GEO

OKseqHMM: a genome-wide replication fork directionality analysis toolkit

Project description:During each cell division, tens of thousands of DNA replication origins are coordinately activated to ensure the complete duplication of the entire human genome. However, the progression of replication forks can be challenged by numerous factors. One such factor is transcription-replication conflict (TRC), which can either be co-directional or head-on and the latter has been revealed as more dangerous for genome integrity. In order to study the direction of replication fork movement and TRC, we developed a bioinformatics tool, called OKseqHMM, to direct measure the replication fork directionality (RFD) as well as replication initiation and termination, along human genome obtained by sequencing of Okazaki fragments (OK-Seq) and related techniques. We have gathered and analyzed OK-seq data from a large number of organisms including yeast, mouse and human, to generate high-quality RFD profiles and determine initiation zones and termination zones by using Hidden Markov Model (HMM) algorithm (all tools and data are available at https://github.com/CL-CHEN-Lab/OK-Seq). In addition, we have extended our analysis to data obtained by related techniques, such as eSPAN and TrAEL-seq, which also contain RFD information. Our works, therefore, provide an important tool and resource for the community to further study TRC and genome instability, in a wide range of cell line models and growth conditions, which is of prime importance for human health.

2022-12-31 | GSE193547 | GEO

DNA molecular combing-based replication fork directionality profiling

Project description:DNA molecular combing-based replication fork directionality profiling

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OKseqHMM: a genome-wide replication fork directionality analysis toolkit

Project description:OKseqHMM: a genome-wide replication fork directionality analysis toolkit

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Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

Project description:The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

| S-EPMC8792808 | biostudies-literature

Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae

Project description:We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication. Examination of the distribution of Okazaki fragments in Saccharomyces cerevisiae strain S288C.

2013-06-18 | E-GEOD-42929 | biostudies-arrayexpress

Characterization of gene expression changes in IRF4-deficient DT40 B cells

Project description:The aim of experiment was to study on genome-wide level IRF4 target genes in chicken DT40 B cell line, by comparizon of gene expression profiles of IRF4-deficient DT40 cells with WT IRF4 DT40 cells .

2017-01-01 | GSE56165 | GEO

Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae

2013-06-18 | GSE42929 | GEO

DT40 genome

Project description:The genome of the DT40 chicken bursal lymphoma cell line

| PRJEB6887 | ENA