DNA replication profiling by BrdU Immunoprecipitation of S.cerevisiae wild-type versus H3K37R mutant
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Namshik Han
PROVIDER: E-MTAB-9097 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Wild-type and isogenic H3K37R yeast cultures were grown in medium containing 2% raffinose as carbone source. Cells were synchronized in G1 with alpha factor. To half culture (100ml), glucose (2% final concentration) and 1.3ml BrdU (50mg/ml stock) and 1.3ml BrdU (50mg/ml stock) was added. To the other half, galactose (2% final concentration) and 1.3ml BrdU (50mg/ml stock). Both flasks were incubated for further 30min at 30C, then cell were collected by centrifugation and resuspended into 100ml of 30C warmed YPA-2%Glucose or YPA-2%Galactose medium containing 25ml HU (2M stock) plus 1.2 ml BrdU (50mg/ml stock) and incubated for 1 hour and 10minutes. YPA-Glucose and YPA-Galactose cultures were immediately transferred to ice/water bath and replication was stopped by addition of NaN3 and cells were processed for DNA immunoprecipitation with anti BrdU antibody.
2021-04-30 | E-MTAB-9999 | biostudies-arrayexpress
Project description:The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells. To investigate relationship between DNA replication and epigenetic modifications
2013-11-05 | E-GEOD-43152 | biostudies-arrayexpress
Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. 5 total experiments with replicates
2015-12-18 | E-GEOD-71050 | biostudies-arrayexpress
Project description:DNA replication profiling by BrdU Immunoprecipitation of S.cerevisiae wild-type versus H3K37R mutant
| PRJEB38412 | ENA
Project description:Nascent strand were purified with either the BrdU or the lambda exonuclease methods from culture human basophilic erythroblasts and sequenced/ Two methods were compared to isolated the Nascent strand, lambda exonuclease digestion or immunoprecipitation after pulse labelling with BrdU The following files were generated by combining round 1 and 2 sample files; FNY01_2_2_Ery_NS_BrdU_macs_summits.bed FNY01_2_2_Ery_NS_BrdU_macs_peaks.bed FNY01_2_2_Ery_NS_lexo_macs_peaks.bed FNY01_2_2_Ery_NS_lexo_macs_summits.bed
2014-05-01 | E-GEOD-50976 | biostudies-arrayexpress
Project description:Nascent strand were purified with either the BrdU or the lambda exonuclease methods from culture human basophilic erythroblasts and sequenced/
2014-05-01 | GSE50976 | GEO
Project description:Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. Newly developed S.cerevisiae chromosome VI tiling array produced by affymetrix was used. Experimental factors:Distribution of various replication and checkpoint related proteins on chromosome VI during S-phase (with or w/o hydroxy urea). Distribution of BrdU incorporated regions on chromosome VI during S-phase with hydroxy urea. Wild type, tof1 deletion mutant, mrc1 deletion mutant, rad9 deletion mutant, sml1 deletion mutant, and sml1 mec1 tel1 triple deletion mutant were also investigated. The results of 74 hybridization files presented in the paper are shown. The type of reference used for the hybridizations, if any: For the analysis of FLAG tagged protein, we usually used Cdc45-3XHA as an internal reference. For the analysis of HA tagged protein, POL1-3XFLAG was usually analyzed simultaneously as a reference. Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. For the analyses of BrdU incorporated regions, anti BrdU antibody bound fraction was compared with SUP (Sup of purified DNA from cells at G1 phase) fraction. During a course of these experiments, we found that S-phase Sup fractions were essentially the same among different genetic backgrounds and did not affect the binding profiles. Based on this observation we use standardized Sups for all experiments. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells. Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus. Or Duplication. The origin of the biological sample: Budding Yeast (Saccharomyces cerevisiae) Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. For the preparation of S-phase cells, cells were released into S-phase without HU and at 16°C collected at the time indicated. These cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest. For the analyses of BrdU incorporation, the same fraction of cells used for ChIP were fixed by ice cold buffer containing 0.1% Azide, and then used for detection of BrdU incorporated regions. Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Young’s lab (http://inside.wi.mit.edu/young/pub/locationanalysis.html). Purified DNA was random primed and amplified by the protocol of Brown’s lab (http://www.microarrays.org). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). For the analysis of BrdU incorporated regions, total DNA from 3X10^8 cells was purified by Qiagen Genomic-tip system (P/N10223, QIAGEN, Germany). DNA was sheared to 300bp by sonication, denatured, and mixed with 2ug of anti-mouse IgG1 dynabeads (P/N110.12, Dynal AS, Norway) bound anti-BrdU monoclonal antibody (2B1D5F5H4E2, MBL, Japan). Immunoprecipitation and purification of antibody bound DNA fraction was carried out following the protocol of Cimbora et al. (Mol. Cel. Biol. 20, 5581-5591, 2000). Purified DNA was random primed and amplified by the Brown’s lab protocol. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling. Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998) The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3’-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA). Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array. In the case of BrdU experiment, loci c6-462 to c6-479 (Ty element) and c6-591 to c6-601 (YFR016c) were excluded from calculation, because those loci gave high background in IP fraction even without BrdU treatment. The software to present the result of discriminant analyses (makeps) is available at our web site. Keywords = DNA replication checkpoint binding profile Keywords: other
2003-08-27 | GSE486 | GEO
Project description:PM01183 on SCLC cell lines BrdU
| PRJNA786938 | ENA
Project description:Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. Newly developed S.cerevisiae chromosome VI tiling array produced by affymetrix was used. Experimental factors:Distribution of various replication and checkpoint related proteins on chromosome VI during S-phase (with or w/o hydroxy urea). Distribution of BrdU incorporated regions on chromosome VI during S-phase with hydroxy urea. Wild type, tof1 deletion mutant, mrc1 deletion mutant, rad9 deletion mutant, sml1 deletion mutant, and sml1 mec1 tel1 triple deletion mutant were also investigated. The results of 74 hybridization files presented in the paper are shown. The type of reference used for the hybridizations, if any: For the analysis of FLAG tagged protein, we usually used Cdc45-3XHA as an internal reference. For the analysis of HA tagged protein, POL1-3XFLAG was usually analyzed simultaneously as a reference. Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. For the analyses of BrdU incorporated regions, anti BrdU antibody bound fraction was compared with SUP (Sup of purified DNA from cells at G1 phase) fraction. During a course of these experiments, we found that S-phase Sup fractions were essentially the same among different genetic backgrounds and did not affect the binding profiles. Based on this observation we use standardized Sups for all experiments. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells. Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus. Or Duplication. The origin of the biological sample: Budding Yeast (Saccharomyces cerevisiae) Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. For the preparation of S-phase cells, cells were released into S-phase without HU and at 16°C collected at the time indicated. These cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest. For the analyses of BrdU incorporation, the same fraction of cells used for ChIP were fixed by ice cold buffer containing 0.1% Azide, and then used for detection of BrdU incorporated regions. Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Youngâs lab (http://inside.wi.mit.edu/young/pub/locationanalysis.html). Purified DNA was random primed and amplified by the protocol of Brownâs lab (http://www.microarrays.org). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). For the analysis of BrdU incorporated regions, total DNA from 3X10^8 cells was purified by Qiagen Genomic-tip system (P/N10223, QIAGEN, Germany). DNA was sheared to 300bp by sonication, denatured, and mixed with 2ug of anti-mouse IgG1 dynabeads (P/N110.12, Dynal AS, Norway) bound anti-BrdU monoclonal antibody (2B1D5F5H4E2, MBL, Japan). Immunoprecipitation and purification of antibody bound DNA fraction was carried out following the protocol of Cimbora et al. (Mol. Cel. Biol. 20, 5581-5591, 2000). Purified DNA was random primed and amplified by the Brownâs lab protocol. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling. Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998) The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3â-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA). Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array. In the case of BrdU experiment, loci c6-462 to c6-479 (Ty element) and c6-591 to c6-601 (YFR016c) were excluded from calculation, because those loci gave high background in IP fraction even without BrdU treatment. The software to present the result of discriminant analyses (makeps) is available at our web site. Keywords = DNA replication checkpoint binding profile Keywords: other
2010-06-05 | E-GEOD-486 | biostudies-arrayexpress
Project description:The purpose of this review is to describe the tools used to detect genome changes, to highlight recent advances in our understanding of large scale chromosome changes that arise in C. albicans and to discuss the role of specific stresses in eliciting these genome changes. The types of genomic diversity that have been characterized suggest C. albicans can undergo extreme genomic changes in order to survive stresses in the human host. We propose that C. albicans and other pathogens may have evolved mechanisms not only to tolerate, but also to generate, large-scale genetic variation as a means of adaptation. Strains included in this series are described in a Eukaryotic Cell review of genomic plasticity of Candida albicans. These strains are well-characterized and some of them are frequently utilized by the Candida community to generate mutant strains. Each strain was analyzed once and all strains were competed against the same reference strain, SC5314 (the first sequenced C. albicans strain).
2010-06-01 | E-GEOD-21643 | biostudies-arrayexpress