Saccharomyces cerevisiae GINS complex progression (Psf2) determined ChIP-chip time course through the cell cycle
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE19818 | GEO | 2010/03/01
SECONDARY ACCESSION(S): PRJNA122141
REPOSITORIES: GEO
Similar Datasets
Project description:N2a cells were transfected with pCAGGS vector or pCAGGS-RABV-M (expressing rabies virus matrix protein) for 36 hours, cells were lysed for SDS-PAGE. The area where the proteins were located in the SDS-PAGE was scooped and cut into small pieces of about 1 mm3; the decolorization solution (15 mM K3Fe(CN)6/50 mM Na2S2O3) was added and shaken until the gelatin block was decolorized, and the supernatant was removed, the decolorization reaction was terminated by adding double-distilled water to submerge the gelatin block, and the washings were aspirated after shaking for 10 min; the gelatin block was submerged with a 50% ACN/100 mM NH4HCO3 ( pH 8.0) solution was used to submerge the gel block, shaking for 10 min, aspirating the washings, and repeating the process for a total of three times; 100% ACN was added to submerge the gel block, shaking for 10 min, aspirating the washings, and then the gel block was dried up in a vacuum extractor; 10 mM DTT/50 mM NH4HCO3 (pH 8.0) solution was added to the gel block, and the gel block was incubated at 56°C for 1h for the reduction reaction, followed by aspirating the The leachate was then added 55 mM iodoacetamide/50 mM NH4HCO3 (pH 8.0) solution, and the reaction was carried out at room temperature in the dark for 30 min, after which the leachate was aspirated; 100% ACN was added, shaken for 20 min, the leachate was aspirated, and the gelatin block was drained; a suitable amount of trypsin was added to the gelatin block, and 50 mM NH4HCO3 solution was added to completely cover the gelatin block Then add 60% ACN/5% formic acid as extraction solution, ultrasonic shaking for 10 minutes, centrifuge and aspirate the supernatant into a new centrifuge tube; repeat the extraction process twice, combine the extraction solution, and drain it in a centrifugal concentrator; desalinate the peptide using a C18 column, and freeze at -20 ℃ to wait for the detection of the machine. Mass spectrometry analyses were performed using a Q Exactive Plus liquid mass spectrometry system from Thermo. Samples were separated by a nanoliter flow rate liquid phase UltiMate 3000 RSLCnano system. Peptide samples were solubilized in the upwelling buffer, inhaled by an autosampler and bound to a C18 capture column (3 μm, 120 Å , 100 μm × 20 mm), followed by elution to an analytical column (2 μm, 120 Å, 75 μm × 150 mm) for separation. An analytical gradient was established using two mobile phases (mobile phase A: 3% DMSO, 0.1% formic acid, 97% H2O and mobile phase B: 3% DMSO, 0.1% formic acid, 97% ACN). The flow rate of the liquid phase was set to 300 nL/min. For mass spectrometry DDA mode analysis, each scan cycle consisted of one full MS scan (R = 70 K, AGC = 3e6, max IT = 20 ms, scan range = 350-1800 m/z) and 15 subsequent MS/MS scans (R = 17.5 K, AGC = 2e5, max IT = 20 ms). AGC = 2e5, max IT = 100 ms). the HCD collision energy was set to 28. the screening window for the quadrupole was set to 1.6 Da. the dynamic exclusion time for repeated ion acquisitions was set to 35 s. Mass spectrometry data generated by Q Exactive Plus were searched by MaxQuant (V1.6.2.10) using the database search algorithm MaxLFQ.The database used for the search was the Mouse Proteome Reference Database.
2023-08-09 | PXD044493 | iProX
Project description:Approximately 3 × 105 NPCs sorted for Sox2-eGFP were mixed with 3 × 104 Drosophila S2 cells per reaction. We performed CUT&RUN using the protocol developed by the Henikoff lab (Skene and Henikoff, 2017). In brief, Bio-Mag®Plus Concanavalin-A (Con A) coated beads (Bangs Laboratories BP531) were washed and activated with Binding buffer. Nuclei of NPCs with S2 spike-in were gently prepared (see Subcellular Protein Extraction section). Activated Con A beads and nuclei were then mixed and rotated for 5 min at room temperature. They were then blocked with Digitonin block buffer for 5 min at room temperature. 0.5-1 μg of primary antibody with 0.25 μg Spike-in antibody (Active Motif 61686) diluted in Digitonin block buffer was added to the bead-nuclei mixture and incubated for 3 hours (histone marks) or O/N (the rest) at 4 °C. Beads were washed three times with Digitonin block buffer and incubated with pA-MNase for 1 hour at 4 °C. Beads were washed three times with Wash buffer and incubated in Wash buffer for 10 min on ice. The pA-MNase was activated by incubating the beads with 2 mM CaCl2 for 25 min on ice and quenched by adding the Stop buffer. DNA was released from the beads by incubating them for 30 min at 37 °C and collected by centrifugation at 16,000 x g for 5 min at 4 °C. DNA was then isolated by using a phenol/chloroform extraction method.
2023-07-12 | GSE212444 | GEO
Project description:ChIP-on-chip of Cdc45 in synchronized cells operating with the analog-sensitive CDK fusion protein (Cdc13-Cdc2). Cells were released from a G2 block or from a G2 block followed by an extended G1 (G1+ 15 min extension). For the G2 block, samples were collected 30 minutes after release. For the G1+15 extension, samples were collected 3 minutes after release. Timepoint of collection was based on qPCR experiments showing Cdc45 binding at origins of replication.
2018-02-04 | GSE108453 | GEO
Project description:A number of methods have been proposed to incorporate multiple scans at different intensities to reduce the quantification error and to minimize effects of saturation, but no direct comparison of their efficacy has been made. Here we used 40 technical replicates to compare individual scans at low, medium and high sensitivity with three methods for combining data from multiple scans (either 2 scan or 3 scan cases) using a variety of metrics Keywords: Multiple Scans
2010-03-22 | GSE19493 | GEO
Project description:Signal intensities of BC-GP array for 105 speimens The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay is an automated microarray-based test, which can detect 12 Gram-positive bacterial genes and 3 resistance determinants using blood culture broths. We investigated signal intensities of microarray spots, and reclassified undetermined results where the automated system failed and various errors were called in blood culture specimens and spiked samples.
2014-05-30 | GSE57245 | GEO
Project description:We addressed the role of cell division during differentiation by blocking the progression of the cell cycle during mesendoderm differentiation. Human pluripotent stem cells (H9 cells) were synchronized in their cell cycle and then induced to differentiate into mesendoderm for 24h. To block the cells in their S phase, we added Hydroxyurea (200 uM) at the beginning of the differentiation process. To block the cells in their G2/M phases, we added nocodazole (100 ng/ml) 8h after the induction of differentiation. At the end of the experiment, the cells were harvested and processed for RNA extraction.
2021-05-31 | GSE130826 | GEO
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
Project description:Background: Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions. This dataset uses different tissue types to explore the consequences of additional sample variance. Keywords: Transcriptional profiling of porcine tissues
2008-02-09 | GSE10446 | GEO
Project description:Time course of after infection (5 min, 20 min and 40 min) of Bacillus subtilis by phage AR9
2017-01-14 | GSE87573 | GEO
Project description:Leaves of Brachypodium distachyon Bd21 were collected at three leaf stage. The total proteins were extracted and then phosphopeptides were enriched by using TiO2 microcolumns and phosphopeptides and their corresponding proteins were identified with LC-MS/MS and Maxquant software. In details, enriched phosphopeptides were separated on a self-packed C18 reverse phase column (75 μm I.D., 150 mm length) (Column Technology Inc., Fremont, CA), which directly connected the nano electrospray ion source to a LTQ-Orbitrap XL mass spectrometer. Pump flow was split to achieve a flow rate at 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% FA (A) and 0.1% FA and 80% ACN (B). A five-step linear gradient of 5% to 30% B in 105 min, 35% to 90% B in 16 min, 90% B in 4 min, 90% to 2% B for 0.5 min and 2% B for 14.5 min was performed. The spray voltage was set to 2.0 kV and the temperature of the heated capillary was 240ºC. For data acquisition, a data-dependent top 10 MS/MS spectraper MS full scan in the Orbitrapanalyser was used. The raw files were processed with Maxquant (version 1.1.1.36) and searched against the NCBI Brachypodium protein database (26,035 entries in total) concatenated with a decoy of reversed sequences. The following parameters were used for database searches: cysteine carbamidomethylation was selected as a fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/MS spectra.
2013-12-17 | PXD000340 | Pride