Dam mutants provide improved sensitivity and spatial resolution for profiling transcription factor binding
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ABSTRACT:
PROVIDER: PRJNA516431 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:DamID, in which a protein of interest is fused to Dam methylase, enables mapping of protein-DNA binding through readout of adenine methylation in genomic DNA. DamID offers a compelling alternative to chromatin immunoprecipitation sequencing (ChIP-Seq), particularly in cases where cell number or antibody availability are limiting. This comes at a cost, however, of high non-specific signal and a lowered spatial resolution of several kb, limiting its application to transcription factor-DNA binding. Here we show that mutations in Dam, when fused to the transcription factor Tcf7l2, greatly reduce non-specific methylation. Combined with a simplified DamID sequencing protocol, we find that these Dam mutants allow for accurate detection of transcription factor binding at a sensitivity and spatial resolution closely matching that seen in ChIP-seq.
2019-01-23 | GSE125447 | GEO
Project description: Not available
| S-EPMC6567924 | biostudies-literature
Project description:We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Keywords: Basal gene expression comparison
2005-07-14 | GSE2928 | GEO
Project description:Stem Cell-Derived Matrices Provide Improved Isolation and Maintenance of hMSC Characteristics
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Project description:Both chromatin access, and binding of AID in B cells are of major scientific interest. Therefore DamID was setup in B cells. AID specific binding could not be identified. In contrast to binding, DAM profiling provide a upmost useful tool in defining chromatin access in vivo.
2016-05-12 | GSE81342 | GEO
Project description:We used Affymetrix microarrays to determine the cisplatin-induced gene expression changes in E. coli deficient in dam and mismatch repair (dam, dam mutS, and mutS mutant strains). E. coli deficient in dam are hypersensitive to cisplatin. However introducing an additional mutation in mismatch repair (i.e., a mutation in mutS or mutL) abrogates this sensitivity and essentially restores wildtype levels of resistance. Experiment Overall Design: Overnight cultures were diluted 1000-fold and grown in Luria-Bertani (LB) medium until the cells reached exponential growth as determined by OD600. The exponentially growing cells were resuspended in M9 minimal medium at a cell density of 2 X 108 cells/ml in a volume of 15 ml and treated with 150 uM cisplatin for 2 hours at 37°C. Following treatment cultures were resuspended in 15 ml LB and allowed to recover for 90 minutes at 37°C. OD600 readings were taken after the recovery period, when RNA isolation began. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) according to the manufactureâs protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20mM 3-[N-morpholino]propanesulfonic acid, 5mM sodium acetate, 1mM ethylenadiaminetetraacetic acide (EDTA)) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure.
2007-07-19 | E-GEOD-2999 | biostudies-arrayexpress
Project description:To determine the transcription start site and the abundance of dam mRNA, we conducted mRNA-specific RNA-Seq experiments. Essentially two strategies were tested. First strategy: We obtained the cDNA with a specific RT-PCR in order to get the mRNA from our dam construct (see DNA-seq of DamID reporter cassette). An enrichment, in which we used a Biotinylated dam oligo and Streptavidin magnetic beads, was needed. Then, similar to SHAPE-seq, the ssDNA ligation was performed with CircLigase. The last step was the PCR reaction in order to prepare the cDNA libraries to be sequenced. Second strategy: Similar to the first one, but the specificity was introduced at the PCR, not at the RT. Thereby, busk cDNA was prepared with random hexamers. The enrichment step was not needed.
2018-03-22 | E-MTAB-5363 | biostudies-arrayexpress
Project description:Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. In the entomopathogenic bacterium Photorhabdus luminescens, a dam ortholog was identified. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.
2017-10-04 | GSE100650 | GEO
Project description:We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Experiment Overall Design: Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further. Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin) in a parallel study (Robbins et al., unpublished results). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturerâs protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.
2007-07-20 | E-GEOD-2928 | biostudies-arrayexpress