RNA-seq of e.coli under growth, regulated growth arrest and disrupted growth arrests - second repetition
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 2000, .
ORGANISM(S): Escherichia coli
SUBMITTER: Adi Rotem
PROVIDER: E-MTAB-14332 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Compares the transcriptomes of E.Coli under natural or unnatural nutrient depletions, leading to either regulated or disrupted growth arrests respectively. E.coli are grown in minimal media with supplemented 0.1% AA. regulated growth arrest (\\"CASP\\"): cultures are grown to stationary phase and sampled after 24 hours in growth arrest. disrupted growth arrest (\\"Disrupted\\"): cultures are grown to ~0.1OD (exponential growth) at which point SHX is added to the growth media. Cultures arrest their growth. Samples are collected 24 hours after growth arrest.
2025-07-01 | E-MTAB-14335 | biostudies-arrayexpress
Project description:RNA-seq of e.coli under growth, regulated growth arrest and disrupted growth arrests - second repetition
| PRJEB78730 | ENA
Project description:Growth inhibition of Rhizobium etli and other organisms by exogenous OA has not been previously reported. We conducted genome-wide transcriptomic analysis to determine the possible mechanism by which OA exerts its growth-inhibitory effect on R. etli CE3. The observed changes fall into several broad categories. First, the decreased expression of genes involved in protein synthesis (including ribosomal genes), replication and energy production indicates that the cells arrest or at least slow their growth, which agrees with the observed growth arrest induced by OA. The upregulation of a key enzyme (PrsAch) for 5'-phosphoribosyl-1'-pyrophosphate (PRPP) synthesis suggests that cells exposed to OA suffer PRPP deprivation. We hypothesized that the PRPP pools were depleted as a result of an increase in the synthesis of OMP stimulated by exogenous OA. PRPP depletion would result in a marked decrease in purine synthesis, which would lead to an imbalance between purine and pyrimidine nucleotides, causing arrest of DNA synthesis and a lack of energetic nucleotides (ADP, ATP). Three independent biological materials with one dyeswap were performed.
2013-10-12 | E-GEOD-46013 | biostudies-arrayexpress
Project description:K562-shX cells are made in an effort to validate TFBS data and ChIP-seq antibodies in Myers lab (GSE32465). K562 cells are transduced with lentiviral vector having Tet-inducible shRNA targeting a transcription factor gene. Cells with stable integration of shRNA constructs are selected using puromycin in growth media. Doxycycline is added to the growth media to induce the expression of shRNA and a red fluorescent protein marker. A successful shRNA cell line shows at least a 70% reduction in expression of the target transcription factor as measured by qPCR. For identification, we designated these cell lines as K562-shX, where X is the transcription factor targeted by shRNA and K562 denotes the parent cell line. For example, K562-shATF3 cells are K562 derived cells selected for stable integration of shRNA targeting the transcription factor ATF3 gene and showed at least a 70% reduction in the expression of ATF3 gene when measured by qPCR. Cells growing without doxycycline (uninduced) are used as a control to measure the change in expression of target transcription factor gene after induction of shRNA using doxycycline. For detailed growth and culturing protocols for these cells please refer to http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. To identify the potential downstream targets of the candidate transcription factor, analyze the mRNA expression profile of the uninduced and induced K562-shX using RNA-seq. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Make K562-shX cells as described in the http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. Measure the mRNA expression levels in uninduced K562-shX and induced K562-shX cells in two biological replicates using RNA-seq. Identify the potential downstream targets of the candidate transcription factor.
2011-12-01 | E-GEOD-33816 | biostudies-arrayexpress
Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This M-bM-^@M-^Xcentromere breathingM-bM-^@M-^Y presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip SAMPLES Origin of each biological sample: Saccharomyces cerevisiae (W303background) Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25M-BM-0C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with M-NM-1-factor (5 M-NM-<g/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from M-NM-1-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35M-BM-0C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 M-NM-<g/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium. EXPERIMENTAL DESIGN Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip). Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples Analysis: ChIP samples were normalized against SUPernatant fractions Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting. Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below: ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65M-BM-:C to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP. Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95M-BM-:C for 10 minutes before being hybridized to the arrays for 16 hours at 42M-BM-:C in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS). ARRAY DESIGN General array design: in situ synthesized arrays from Affymetrix Location and ID of each spot on arrays: available upon request from Affymetrix Probe type: oligonucleotide Availability of arrays: commercially available from Affymetrix S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636 S. cerevisiae T iling Array (Forward), P/N# 520286 REFERENCES Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20 Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083 Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578 Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799 Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386
2007-09-29 | E-GEOD-8661 | biostudies-arrayexpress
Project description:K562-shX cells are made in an effort to validate TFBS data and ChIP-seq antibodies in Myers lab (GSE32465). K562 cells are transduced with lentiviral vector having Tet-inducible shRNA targeting a transcription factor gene. Cells with stable integration of shRNA constructs are selected using puromycin in growth media. Doxycycline is added to the growth media to induce the expression of shRNA and a red fluorescent protein marker. A successful shRNA cell line shows at least a 70% reduction in expression of the target transcription factor as measured by qPCR. For identification, we designated these cell lines as K562-shX, where X is the transcription factor targeted by shRNA and K562 denotes the parent cell line. For example, K562-shATF3 cells are K562 derived cells selected for stable integration of shRNA targeting the transcription factor ATF3 gene and showed at least a 70% reduction in the expression of ATF3 gene when measured by qPCR. Cells growing without doxycycline (uninduced) are used as a control to measure the change in expression of target transcription factor gene after induction of shRNA using doxycycline. For detailed growth and culturing protocols for these cells please refer to http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. To identify the potential downstream targets of the candidate transcription factor, analyze the mRNA expression profile of the uninduced and induced K562-shX using RNA-seq. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2011-12-01 | GSE33816 | GEO
Project description:Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm (dm4) results in attenuated endoreplication and growth arrest early in larval development. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis. Drosophila larvae were collected 24 hours after egg deposition. Sample comparisons were performed using 3 biological independent experimental replicates of dm4 that were each compared to a common reference (wild type larvae of the same genetic background as dm4). For each comparison, a dye-swapped technical replicate was also performed and the paired results were averaged and used as a single observation.
2010-05-26 | E-GEOD-9729 | biostudies-arrayexpress
Project description:Campylobacter jejuni is a human pathogen which causes campylobacteriosis, one of the most widespread zoonotic enteric diseases worldwide. Growth of Campylobacter can be improved through the addition of glutamine to media which serves as the nitrogen source. RNAseq was used to identify the transcriptomic response of Campylobacter jejuni when the nitrogen source was switched from serine (poor growth) to glutamine (good growth) in chemostat cultures.
2022-12-01 | E-MTAB-11495 | biostudies-arrayexpress
Project description:We constructed a tiling microarray, covering nearly all of the intergenic regions larger than 50 bp on both strands of the genome of the marine picocyanobacterium Synechococcus WH7803. We analyzed transcript levels from cultures grown under ecologically relevant stress conditions. The investigated stress conditions were cold stress, high light stress, phosphate depletion and iron depletion. We identified several previously unknown small RNAs, partially differentially expressed. The detected RNAs provide a starting point for further investigations on the acclimatisation to different stresses for Synechococcus WH7803. For every applied growth condition the cultures were grown in triplicates as were the respective controls. Respective controls were treated the same as the stressed bacterial cultures in terms of centrifugation and / or dilution. Bacteria were harvested by rapid filtering and directly freezed by liquid nitrogen.
2012-03-30 | E-GEOD-28263 | biostudies-arrayexpress