Mapping of genome-wide DSBs in HEK293T cells using the RAFT procedure.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE53811 | GEO | 2015/03/05
SECONDARY ACCESSION(S): PRJNA233403
REPOSITORIES: GEO
Similar Datasets
Project description:Eukaryotic chromosomes are subjected to spontaneous fragmentation even under quick isolation of DNA in a solid phase by strong treatment with 0.1 M EDTA, 1% SDS and proteinase K (1 mg/ml). The long DNA fragments of excised chromosomal DNA were denoted as forum domains. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). We performed a genome-wide mapping of DSBs in human HEK293T cells cultured cells using Illumina deep sequencing of the termini of forum domains. We found that in rDNA units the hot spots of DSBs are distributed non-randomly. Mostly they are located in IGS. Genome-wide mapping of DNA DSBs in HEK293T cells
2014-10-04 | E-GEOD-49302 | biostudies-arrayexpress
Project description:Eukaryotic chromosomes are subjected to spontaneous fragmentation even under quick isolation of DNA in a solid phase by strong treatment with 0.1 M EDTA, 1% SDS and proteinase K (1 mg/ml). The long DNA fragments of excised chromosomal DNA were denoted as forum domains. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). We performed a genome-wide mapping of DSBs in human HEK293T cells cultured cells using Illumina deep sequencing of the termini of forum domains. We found that in rDNA units the hot spots of DSBs are distributed non-randomly. Mostly they are located in IGS.
2014-10-04 | GSE49302 | GEO
Project description:For genome-wide identification and analysis of endogenous DSBs in HEK293T cells chromosomes the ligation-mediated PCR and deep sequencing of the DSBs library were used. The cells were included in low-melt agarose, and the plugs were used for isolation of DNA in a solid phase using incubation in the solution containing 0.5 M EDTA (pH 9.5), 1% sodium laurylsarcosine, and 1-2 mg of proteinase K per ml for 40-48 hr at 50°C. Then DNA was isolated by electro-elution and used for ligation with biotinylated ds-adapter. To shorten large DNA molecules the digestion with Sau3A endonuclease was performed. Then the short regions of DNA attached to DSBs sites were selected using the streptavidin magnespere paramagnetic particles. To prepare samples for amplification of DSBs library the Sau3A adapter was ligated at the opposite to DSBs sites. PCR amplification was performed using the primers corresponding to biotinylated and Sau3A adapters. The library was used for paired end sequencing. The corresponding reads were processed and used for analysis of their distribution in human genome.
2022-07-15 | GSE201829 | GEO
Project description:Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. We performed a genome-wide mapping of forum domains termini in human HEK293T cells cultured cells using deep sequencing of the termini. We found that forum domains termini correspond to the fragile sites in human chromosomes and forum domains contain clusters of several or many genes inside. The largest forum domains correspond to the coordinately expressed main clusters of HOX genes genes. Our results indicate that forum domains correspond to big multi-gene chromosomal units some of which could be co-coordinately activated or repressed. 2 sample examined: forum termini from HEK293T cells in two independent experiments
2013-03-31 | E-GEOD-35065 | biostudies-arrayexpress
Project description:Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. We performed a genome-wide mapping of forum domains termini in human HEK293T cells cultured cells using deep sequencing of the termini. We found that forum domains termini correspond to the fragile sites in human chromosomes and forum domains contain clusters of several or many genes inside. The largest forum domains correspond to the coordinately expressed main clusters of HOX genes genes. Our results indicate that forum domains correspond to big multi-gene chromosomal units some of which could be co-coordinately activated or repressed.
2013-03-31 | GSE35065 | GEO
Project description:Chromatin undergoes major remodeling around DNA double strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high resolution map of gammaH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit from the cohesin complex, previously characterized as required for repair by homologous recombination. We found that the recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs. In addition, we show that the cohesin complex, which was also recently proposed to be a key player in chromosome organisation and chromatin looping, controls gammaH2AX distribution within domains. Indeed, as we reported for transcription, cohesin binding antagonizes gammaH2AX spreading. Remarkably, depletion of cohesin leads to an increase of gammaH2AX at cohesin-bound genes (revelead by gammaH2AX mapping in upon SCC1 siRNA), associated with a decrease in their expression level after DSB induction. Thus our study identifies a novel role for the cohesin complex in protecting the genes located in gammaH2AX domains from both gammaH2AX spreading and transcriptional shut-down after DSB induction.
2012-01-01 | E-TABM-1164 | biostudies-arrayexpress
Project description:In this study we developed TRS, a computational approach to determine the 3’ termini of transcripts from total RNA data generated by the RNAtag-seq protocol. To show the applicability of our approach, we applied our algorithm to sequencing data generated by the RNAtag-seq and the term-seq protocols. In term-seq, a 3’ adaptor is ligated to the RNA 3' termini prior to the RNA fragmentation, and thus, the sequences adjacent to the adapter sequence in the library reads represent original 3' termini that were present in the RNA sample. The RNAtag-seq protocol involves ligation of a 3’ adaptor after the RNA fragmentation and thus, the sequences adjacent to the adapter sequence in the library reads represent genuine 3’ termini that were present in the RNA sample and artificial 3’ termini generated by the RNA fragmentation. We show that our computational approach can reliably identify genuine 3’ termini from RNAtag-seq data by comparing the set of identified 3’ termini based on the RNAtag-seq and the term-seq protocols.
2023-07-15 | E-MTAB-12429 | biostudies-arrayexpress
Project description:Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. <br><br>We performed a genome-wide mapping of forum domains termini in Drosophila Schneider 2 cultured cells using Drosophila melanogaster genomic tiling microarray consisting of overlapping 3 kb fragments. We found that forum domains contain clusters of several or many genes inside. The largest forum domains correspond to the coordinately expressed main clusters of homeotic genes inside BX-C and ANTP-C, cluster of histone genes and clusters of piRNAs. PRE/TRE and binding sites of transcription factors are often reside inside domains, and do not overlap with forum domains termini. The terminal regions of domains often correspond to chromosomal sites containing different classes of mobile elements, binding sites of SuUR protein and regions of intercalary heterochromatin. Our results indicate that forum domains correspond to big multi-gene chromosomal units some of which could be co-coordinately activated or repressed.
2010-11-01 | E-MEXP-2468 | biostudies-arrayexpress
Project description:Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (Kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops.
2018-04-24 | GSE98161 | GEO
Project description:RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5'-PO4 and 3'-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as the first human enzyme promoting RNA ligation between 5'-PO4 and 3'-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. This data provides the groundwork for establishing a human RNA repair pathway.
2023-01-27 | PXD038132 | Pride