Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Tiling path analysis of seven temperature sensitive S. cerevisiae BY4741 mutants

ABSTRACT: Seven temperature sensitive Saccharomyces cerevisiae BY4741 mutants (rsc3-1, abf1-101, reb1-212, rap1-1, mcm1, tbf1 and cep3-1) were grown at restrictive temperatures until a difference in OD600 was observed relative to a wild-type control. Nucleosomal DNA, whole genomic DNA and total RNA were isolated and hybridized onto yeast whole-genome tiling arrays.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Timothy Hughes

PROVIDER: E-MEXP-1754 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A library of yeast transcription factor motifs reveals a widespread function for Rsc3 in targeting nucleosome exclusion at promoters.

Badis Gwenael G Chan Esther T ET van Bakel Harm H Pena-Castillo Lourdes L Tillo Desiree D Tsui Kyle K Carlson Clayton D CD Gossett Andrea J AJ Hasinoff Michael J MJ Warren Christopher L CL Gebbia Marinella M Talukder Shaheynoor S Yang Ally A Mnaimneh Sanie S Terterov Dimitri D Coburn David D Li Yeo Ai A Yeo Zhen Xuan ZX Clarke Neil D ND Lieb Jason D JD Ansari Aseem Z AZ Nislow Corey C Hughes Timothy R TR

Molecular cell 20081201 6

The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes. One-third of the binding specificities have not been previously reported. Several binding sequences have striking genomic distributions relative to transcription start sit ...[more]

PMID: 19111667

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Project description:Limb buds were dissected from E10.75 mouse embryos, fixed in 1% formaldehyde for 15 min at room temperature, washed 3 times with cold PBS and stored at -800C. Pools of 16 limb buds were used for each ChIP-chip experiment. ChIP was performed according to (Lee et al., 2006) using 2 ?g of anti-CTCF antibodies (A300-543A, Bethyl Laboratories) and EZview Red Protein G/A Affinity Gel (Sigma). Immunoprecipitated and whole cell extract DNA (input) were treated with RNaseA, proteinase K and purified by 2 rounds of extraction with phenol:chloroform: isoamyl alcohol. ChIP and input DNA were amplified using Ligation-Mediated PCR (Lee et al., 2006). PCR was limited to 15 cycles. 1.5 ?g of ChIP and input DNA were fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to the Chromosome 2 and X tiling arrays (Affymetrix). Tiling arrays data were quantile normalized within cDNA/genomic DNA or ChIP/ input replicate groups using R packages, STARR and Ringo (Zacher et al., 2010; Toedling et al., 2007). The ratio of probe intensity between the experiment and the control groups were computed considering the median values over replicates. The ratios were smoothened by computing the running medians with a half window size set to 150 bp and a minimum of 5 probes per window. To identify enriched regions a minimum of 3 consecutive probes with a smoothed ratio exceeding a threshold have been considered. The threshold has been fixed by taking the 99th percentile of the estimated null distribution of the ratios. Only ChIP enriched regions with score log2?1.5 and width ?150 bp were considered for further analysis. As a complement, array data were quantile normalized within ChIP/input replicate groups and scaled to medial feature intensity of 500 using TAS software (Affymetrix). For each genomic position, a dataset was generated consisting of all probes mapping within a sliding window of 250 bp. The averaged ratios were plotted along the genomic DNA sequence using Integrated Genome Browser (IGB) software (Affymetrix).

Project description:One day-old broilers (Ross/Ross, Longenecker's Hatchery, Elizabethtown, PA) were housed in Petersime starter brooder units and randomly assigned to 4 groups (5 birds/group). Carvacrol (Nature Chemicals, Hamburg, Germany) and cinnamaldehyde (Alys Technologies SA, Bussigny-près-Lausanne, Switzerland) were obtained from the compound obtained from synthesis. Crushed C. annuum fruits (Pushp Brand Spices, Munimji Foods and Spices Pvt. Ltd. Indore, India) were extracted with volatile solvents using the percolation method to obtain oleoresin. Chickens were fed for 7 days beginning from hatch with a standard diet alone (control) or with diets supplemented with carvacrol, cinnamaldehyde, or Capsicum oleoresin.<br> Following euthanization of the birds, intestines were cut longitudinally and washed three times with ice-cold Hanks? balanced salt solution (HBSS) containing 100 U/ml of penicillin and 100 mg/ml of streptomycin (Sigma, St. Louis, MO). The mucosal layer of entire intestine was carefully scraped using a surgical scalpel and intraepithelial lymphocytes (IELs) were isolated by Percoll density gradient centrifugation as previously described (Min et al. 2005). Total RNA was isolated from 5.0 x 107 cells using Trizol (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini RNA Purification Kit (Qiagen, Valencia, CA). Aminoallyl-labeled RNA from IELs was prepared using the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion, Austin, TX). Two of 20 ?g aliquots of each aminoallyl-RNA sample were fluorescently labeled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen) and labeled RNAs were column-purified using the RNA Amplification Kit (Ambion). RNA concentrations and labeling efficiencies were determined spectrophotometrically.<br> Hybridizations were performed using HybIt hybridization buffer (TeleChem, Sunnyvale, CA) in ArrayIt reaction cassettes at 50°C overnight as described (Kim et al., 2008). After hybridization, the slides were rinsed in 0.5 X SSC, 0.01% SDS at room temperature and washed once for 15 min in 0.2 X SSC, 0.2% SDS at 50°C, 3 times for 1 min in 0.2 X SSC at room temperature, and 3 times for 1 min in distilled water at room temperature. Each sample had a repeated hybridization using the alternate fluorescent dye between the treatment and control.

Dataset Information

Tiling path analysis of seven temperature sensitive S. cerevisiae BY4741 mutants

Publications

A library of yeast transcription factor motifs reveals a widespread function for Rsc3 in targeting nucleosome exclusion at promoters.

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