Browse
Submit Data
Databases
API
Help

Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

100 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Massive sequencing of hybrid DNA obtained by DNA:RNA immunoprecipitation (DRIP) in extracellular microvesicles (MV) produced by HeLa cells (using S9.6 antibody)

ABSTRACT: Hybrid (Bound) and non-hybrid (Unbound) DNA from Hela EV was isolated using DRIP procedure as follows. Between 1-5μg of nucleic acid DNA from EVs (DNase0 treated) was incubated overnight with 1μg of anti-DNA-RNA Hybrid (S9.6) antibody (KeraFast) under rotation at 4°C in 500μl of binding buffer (10mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The following day, 25μl of A/G magnetic beads (Pierce™) were washed twice in 500μl of binding buffer (30' min each wash) and added for 2h at room temperature under rotation to the S9.6+nucleic acid complex. The nucleic acids bound to the S9.6 antibody (Hybrid fraction or Bound) were separated from those unbound (Non-Hybrid or Unbound) by using a magnetic rack (which captured the Bead+Hybrid fraction). 500μl of the unbound fraction (bead-free) were transferred into a new 1.5ml tube and collected for further analysis. The Bead+Hybrid fraction was washed twice in 250μl binding buffer for 15' min at room temperature under rotation. The hybrids were then eluted with 250μl of elution buffer (50mM TRIS pH 8.0, 10mM EDTA, 0.5% SDS) for 15' min at room temperature under rotation. The elution step was repeated twice to make sure that all the hybrid molecules were collected. As a control of all there experiments to make sure, we were isolating and precipitating Hybrid DNA:RNA molecules, samples were also pre-treated with RNaseH1 (RNaseH), which degrades the RNA filament only when present in the hybrid structure.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Homo sapiens

SUBMITTER: Maurizio Cardelli

PROVIDER: E-MTAB-9634 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Similar Datasets

Pericentromeric satellite repeat expansions through RNA-derived DNA intermediates in cancer

Project description:Aberrant transcription of the HSATII pericentromeric satellite repeat is present in a wide variety of epithelial cancers. We observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under non-adherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generate complementary DNA intermediates in the form of DNA:RNA hybrids. Single molecule sequencing of tumor xenografts shows that HSATII RNA-derived DNA (rdDNA) molecules are stably reincorporated within pericentromeric loci, leading to progressive expansion of these regions We analyzed the dynamics of HSATII RNA and DNA level changes using single molecule sequencing (Helicos/SeqLL) in SW620 colon cancer cells transitioned from 2D in vitro culture conditions to growth as mouse xenografts and vice versa.

2015-11-02 | E-GEOD-64471 | biostudies-arrayexpress

RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships RDIP-seq)

Project description:Mapping of RNA:DNA hybrids in human cells reveals a number of characteristics of these non-canonical nucleic acid structures. A directional sequencing approach reveals the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to the stability of these structures. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation. Studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation. Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting a model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo. Investigation of locatization of RNA in RNA:DNA hybrids genome-wide in HEK293T and IMR-90 cells through RNA:DNA hybrid immunorprecipitation strand-specific sequencing

2015-06-15 | E-GEOD-68948 | biostudies-arrayexpress

MiRNome profiling of glioblastoma tissues and peritumoral regions

Project description:Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor. Its prognosis is inexorably unfavorable, as these tumors drive affected patients to death usually within 15 months after diagnosis (short term survivors, ST), with the only exception of a small fraction of patients (long term survivors, LT) surviving longer than 36 months. Even after the frontline therapeutic approach, including surgical resection followed by chemo- and radiotherapy, the cause of death in most cases is tumor recurrence, which occurs in peritumoral tissues in about 95% of patients. Here, we provide a comprehensive molecular analysis of a set of ST and LT samples derived from frankly tumoral areas (C) and from the peritumoral regions (P) of the same patients. By performing microRNA deep sequencing, we collected data showing that P areas differ from healthy white matter, but share with C samples, a number of microRNAs

2017-03-08 | E-MTAB-3239 | biostudies-arrayexpress

Small RNAs as important regulators for the hybrid vigour of super-hybrid rice

Project description:Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis. We constructed six sRNA sequencing libraries and six mRNA sequencing libraries of flag leaves and panicles of the super-hybrid rice Liangyou-pei9 (LYP9) combination at the grain-filling stage. The above hybrid rice combination includes F1 hybrid LYP9 and its parental lines including the male-sterile line Peiai64s (PA64s) and the restorer line 93-11.

2014-08-17 | E-GEOD-51468 | biostudies-arrayexpress

Dual RNA-seq of M. tuberculosis H37Rv infecting macrophage-like THP-1 cells during 48h

Project description:Transcriptional changes during early infection of macrophage-like THP-1 cell line with pathogenic bacterium Mycobacterium tuberculosis. RNAseq samples were taken at 0h (THP-1 cells growing in the RPMI medium), and after 4h, 24h and 48h post infection. Bacterial enrichment was performed to increase the amount of bacterial mRNA in the samples. Non-enriched samples were used to map THP-1 cells transcripts; enriched samples were used to map M. tuberculosis transcripts the corresponding genomes.

2017-08-01 | E-MTAB-5287 | biostudies-arrayexpress

Comparative transcriptomic analysis of human and Drosophila extracellular vesicles reveals extensive conservation

Project description:Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that Drosophila, like human cells release exosome-like EVs with diameter ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNA pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these extensive similarities suggest that EVs could also enable RNA-mediated intercellular communication in Drosophila. We performed RNA-seq on extracellular vesicles purified from of human and Drosophila cell line cultures. S2R+ and D17 Drosophila EVs were analyzed, along with human A431 and HepG2 EVs. No ribosomal RNA depletion or polyA selection was performed on EV samples. For comparative analyses, we also analyzed total cellular RNA from Drosophila D17 and human HepG2. Ribodepletion was performed on cellular samples.

2016-06-15 | E-GEOD-76173 | biostudies-arrayexpress

Transcriptome and methylome interactions in rice hybrids

Project description:This research reports genome-wide measurements of genetic and epigenetic patterns of inheritance through an integrative analysis of BS-seq, RNA-seq, and siRNA-seq data in two inbred parents of the Nipponbare (NPB) and Indica (93-11) variety of rice and their hybrid offspring. We generated integrative maps of whole genome cytosine methylation profiles (BS-Seq), transcriptional profiles (RNA-seq), and small RNA profiles (sRNA-seq) to characterize two rice subspecies, Oryza sativa spp japonica (Nipponbare) and Oryza sativa spp indica (93-11) and their two reciprocal hybrid offspring using Illumina's sequencing-by-synthesis (SBS) platform .

2012-07-10 | E-GEOD-38480 | biostudies-arrayexpress

GA20-oxidase overexpression and heterosis stimulate complementary growth mechanisms

Project description:This experiment involves RNAseq data on 3 parental lines and 2 hybrid crosses. The parents: inbred lines B104 and Mo17, and the GA20OX transgenic line in B104 background. The hybrid crosses: B104 x Mo17, GA20OX x Mo17.

2021-11-17 | E-MTAB-11102 | biostudies-arrayexpress

Plasticity of Mitochondrial DNA Inheritance and its Impact on Nuclear Gene Transcription in Yeast Hybrids grown on YP Glycerol 28C

Project description:Mitochondrial DNA (mtDNA) in budding yeast is biparentally inherited, but colonies rapidly lose one type of parental mtDNA, becoming homoplasmic. Therefore, hybrids between different yeast species possess two homologous nuclear genomes, but only one type of mitochondrial DNA. We hypothesise that the choice of mtDNA retention is influenced by its contribution to hybrid fitness in different environments, and that the allelic expression of the two nuclear sub-genomes is affected by the presence of different mtDNAs in hybrids. Here, we crossed Saccharomyces cerevisiae with S. uvarum under different environmental conditions and examined the plasticity of the retention of mtDNA in each hybrid.

2020-04-16 | E-MTAB-7922 | biostudies-arrayexpress

Plasticity of Mitochondrial DNA Inheritance and its Impact on Nuclear Gene Transcription in Yeast Hybrids Compared to parents strains

2020-10-26 | E-MTAB-8900 | biostudies-arrayexpress