Half-Half experiments
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE46932 | GEO | 2016/05/12
SECONDARY ACCESSION(S): PRJNA203103
REPOSITORIES: GEO
Similar Datasets
Project description:These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjees El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Strain Names: AIIIpO139#1 / AIIIpO139#3 / AIIIpO139#4 / AIIIpO139#5 are four clones analyzed after the second experiment; AIVpO139#2 / AIVpO139#4 / AIVpO139#5 / AIVpO139#8 are four clones analyzed after the fourth independent experiment. Two MA replicates for each clone were done. A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's. Keywords: all_pairs, array CGH
2007-04-26 | GSE5933 | GEO
Project description:RNA decay was measured in Prochlorococcus after inhibition of transcription by rifampicin using customized Affymetrix gene expression arrays. RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. Using a combination of microarrays, quantitative RT-PCR and a new algorithm for determining RNA decay rates, we found a median half-life of 2.4 min and a median decay rate of 2.6 min for expressed genes â two-fold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (ca. 18 min) were for highly expressed genes. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. We hypothesize that the fast turnover relative to generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will inform on the modelling of cell processes and help interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms. Prochlorococcus cells were treated with rifampicin, which prevents initiation of new transcripts. Cells were harvested at 0 min (before rifampicin addition), 2.5 min, 5 min, 10 min, 20 min, 40 min and 60 min after rifampicin addition.
2010-05-16 | E-GEOD-17075 | biostudies-arrayexpress
Project description:Background We have developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse ES cells, this retrovirus-based method generated deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drugs-selected clones. Importantly, several of engineered ES cell clones, mostly those with large deletions, showed major alteration in cell fate. The set of complementary replication-defective retroviruses exploited the Cre-loxP recombination system and reconstitution of a functional neomycin cassette for selection of recombination events. The first loxP sequenced was delivered using the anchor virus A1. The integration was selected on puromycin. ES cell clones containing one copy of the virus A1 were isolated and expanded (primary clones). A second loxP was introduced by retroviral gene transfer using the saturation virus S1, selected on hygromycin (secondary clones). The transient expression of the recombinase Cre in secondary clones allowed the recombination between the loxP sites and the functional reconstitution of a split neomycin expression cassette. Neomycin resistant clones (tertiary clones) were isolated and expanded. Chromosomal deletions are expected to have occurred in neomycin resistant clones that have lost both puromycin and hygromycin resistance genes. In addition, other chromosomal rearrangements (e.g., inversions, translocations, etc.) are achievable using this system. Aim Inverse-PCR, array-based comparative genomic hybridization (aCGH) and spectral karyotyping were employed to confirm deletions (or other Cre-induced rearrangements) in several tertiary clones and to assess the genomic integrity of the ES cells altered. Conclusions aCGH conclusions are summarized together with complementary results from inverse-PCR and spectral karyotyping in 3 tables: Table 1: Summary of the virus A1 integration Table 2: Characteristics of independent deletions confirmed by IPCR-aCGH Table 3: Confirmed or suspected recombination events in trans Notes for Table 2: Mapping and deletion analyses were done using the UCSC Genome Browser (NCBI mouse Build 33). {a} Tertiary clones are labeled according to their family number (same integration of virus A1), followed by a specific id number. If more than one clone presented a redundant rearrangement within the same group infected with virus S1, only one is reported for clarity. {b} Anomaly that was not present in the primary clone from which the tertiary clone was derived, as determined by aCGH or SKY. ( -), no anomaly; (+), additional anomaly. {c} The deletion is not observed, in agreement with the resolution of aCGH. {d} Normal except for the loss of chromosome Y. {e} Amplification of chromosome 1. {f} Amplification of chromosome 8. {g} Many chromosomes were lost according to SKY. {h} Amplification on chromosome 14. Id, identification; kb, kilobase pairs; no., number; aCGH, array-based comparative genomic hybridization; SKY, spectral karyotyping; I-PCR, inverse-PCR; n.d., not determined. Keywords: comparative genomic hybridization
2007-01-13 | GSE6706 | GEO
Project description:A procedure to acquire two Multiple-Quantum Magic Angle Spinning (MQMAS) NMR experiments with the same instrument time is presented. A triply tuned probe is utilized with multiple receivers to collect data with staggered acquisitions and thus more efficiently use the instrument time. The data for one nucleus is collected during the recovery delay of the other nucleus, and vice versa. The instrument time is reduced to 60-80% of the time needed for the single acquisition collection Specifically our approach is presented for recording triple-quantum (3Q) 17O and either 3Q or quintuple-quantum (5Q) 27Al MAS NMR spectra of a 1.18Na2O•5SiO2•Al2O3 glass gel.
| S-EPMC7661836 | biostudies-literature
Project description:Due to the lack of controlled microarray experimental data, evaluation of gene identification methods has been performed using simulated data. As the ability of these approaches to mimic actual experimental data is questionable, conclusions drawn can be misleading. We applied spike-in approaches to develop experimental benchmark data and used it to evaluate various gene identification methods. mRNA that correspond to specific spots on a microarray were obtained by in-vitro transcription of selected clones. They were then spiked in at 11 varying concentrations to a common pool of mRNA, creating a set of known differentially expressed genes. mRNA was obtained from mouse ATCC CRL1606 hybridoma cells. From self hybridization results 200 genes that consistently exhibited a log ratio value close to 0 were randomly selected across a range of intensity. In-vitro transcripts of these 200 genes were obtained from their respective clones. From RNA gel electrophoresis and test array results, 169 of the 200 transcripts were deemed successful. Experiments were then conducted by spiking these 169 transcripts to the common pool of mRNA extracted from the hybridoma cells. With exception of the spiked in genes, the experimental set-up is identical to a self hybridization. This experiment is thus different from other microarray experiments as the set of differentially expressed genes is known as they have been artificially created. Spiked in genes were introduced at 11 concentrations: 0pmol (self hybridization), 0.025pmol, 0.05pmol, 0.10pmol, 0.15pmol, 0.20pmol, 0.25pmol, 0.5pmol, 0.75pmol 1pmol and 2pmol. For concentrations 0pmol, 0.05pmol, 0.10pmol, 0.25pmol, 0.50pmol and 0.75pmol, 6 replicates were obtained. For concentrations 0.025pmol, 0.15pmol, 0.20pmol, 1pmol and 2pmol, 3 replicates were obtained. Dye swaps were not performed for these experiments. Keywords: other
2005-05-03 | GSE2548 | GEO
Project description:Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induced chromosome mis-segregation in non-transformed RPE1-hTERT cells and derived multiple stable clones with various degrees of aneuploidy. We performed an unbiased genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogated their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activated the DNA damage response, and were consequently more resistant to further DNA damage induction. Aneuploid cells also exhibited elevated RAF/MEK/ERK pathway activity, and were more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitized aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. These results were validated in human cancer cell lines. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, revealing a novel therapeutically-relevant cellular dependency of aneuploid cells. This submission contains the raw files, the library used for the analysis, and the corresponding DIA-NN report and associated files.
2024-07-08 | PXD048833 | Pride
Project description:RNA decay was measured in Prochlorococcus after inhibition of transcription by rifampicin using customized Affymetrix gene expression arrays. RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. Using a combination of microarrays, quantitative RT-PCR and a new algorithm for determining RNA decay rates, we found a median half-life of 2.4 min and a median decay rate of 2.6 min for expressed genes – two-fold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (ca. 18 min) were for highly expressed genes. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. We hypothesize that the fast turnover relative to generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will inform on the modelling of cell processes and help interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms.
2009-12-31 | GSE17075 | GEO
Project description:The abf1-1 mutant and wild type cells were grown at 30 degrees C in YPAD until OD600 reached 1.0-1.2. The same volume of 42 degrees C preheated YPAD was added for each flask. After 45 min incubation at 36 degrees C, cells were harvested and RNA was isolated. Four repeats of microarray results are in this series. Keywords: repeat sample
2004-06-23 | GSE1492 | GEO
Project description:We calculated global RNA half lives for all genes in the cyanobacteria Synechococcus sp. strain PCC 7002. Samples from three replicates of the WT strain were taken before and following the addition of the antibiotic rifampicin to stop nascent transcription. RNA spike-ins were added for normalization and using the number of RNA-sequencing reads we were able to calculate half lives for genes, transcripts, and for each position on the genome.
2019-01-01 | GSE109174 | GEO
Project description:Human embryonic stem (ES) cell lines acquire recurrent karyotypic abnormalities that may promote growth and survival in long-term culture. Cells with these abnormalities are often present at low levels in human ES cell cultures, requiring subcloning for isolation and analyses. We have developed a rapid method to isolate either abnormal or normal clones from karyotypically mosaic human ES cell cultures that we call Genetic Diagnosis at Passage (G-DAP). We have used this technique to isolate three distinct abnormal sublines from H9 human ES cells in continuous culture. These sublines were characterized by G-banding, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and array comparative genomic hybridization (aCGH). One subline (H9.DT) has, as it’s only abnormality, a duplication of 12p capped by a 5q subtelomere. A related subline (H9.TSF) has an additional copy of the derivative chromosome 12, partial trisomy of 17q, and trisomy 14. The third subline (H9.DE) has an interstitial deletion of 18q as the sole anomaly. Despite these karyotypic abnormalities, all three sublines retain markers of the undifferentiated state. The subline H9.TSF demonstrated the ability to form embryoid bodies consisting of all three germ layers. Significantly, H9.DT and H9.TSF sublines show a classic karyotype progression that mirrors that seen in both hematopoietic and solid tumors. This progression was accompanied by an impressive increase in growth potential. The isolation of human ES cell sublines with clonal cytogenetic abnormalities provides a valuable tool for study of factors that promote in vitro genetic changes and for analysis of mechanisms of aneuploidy related to genetic progression found in human cancers.
2008-07-18 | GSE12119 | GEO