Measuring expression from randomly-designed DNA in S. cerevisiae
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE47004 | GEO | 2013/09/01
SECONDARY ACCESSION(S): PRJNA203264
REPOSITORIES: GEO
Similar Datasets
Project description:Here, we integrated essentially random synthetic DNA sequences into the yeast genome (HO locus) and measured the expression via custom Agilent tiling array. The purpose of this was to compare the predictions of where transcription would occur output by our computational model to the expression observed in the cell. The four constructs (A1B1, A1B2, A2B1, A2B2) were integrated into S. cerevisiae, cells were grown and from these both gDNA and mRNA were isolated. G-coupled dyes were used to label samples with Cy3 for mRNA and Cy5 for gDNA. Samples were sheared/hydrolyzed to an average size of about 250bp, hybridized to the array, and quantified. The GFF files include features of the region tageted for tiling array analysis in gff format (https://genome.ucsc.edu/FAQ/FAQformat.html#format3). These include the locations of genomic features (ORFs, transcripts) as well as the locations of the inserts, and the KanMX selectable marker. microarray_normalized_log+.wig and microarray_normalized_log-.wig include the tiling array data across each of the four inserts in wig format (https://genome.ucsc.edu/goldenPath/help/wiggle.html) for the top and bottom DNA strands, respectively. This data are provided on a log scale and were normalized as follows: First, we normalized the data using spacial detrending of the microarray slide. Next, we normalized the tiling array data using a background model (HUBER et al. 2006) which relates, for each probe (k), the signal intensity (yk) to the background signal (Bk) plus the amount of nucleic acid in the sample (xk) times the proportionality factor (ak), or yk=Bk+xk*ak. Here, we want to determine the amount of nucleic acid in the sample (xk). We modified this normalization protocol to take advantage of the fact that the target sequences of many probes were absent from some samples (e.g. the A1B1probe target sequences are absent from the A2B2 strain). Here, we estimated separate mRNA and gDNA background signals (Bk) for each probe by taking the median signal intensity of the probe in strains lacking the target DNA sequences. We estimated the proportionality factor for each probe (ak), corresponding to how probe intensity changes with the amount of nucleic acid, by comparing the background-normalized gDNA signal at each probe to the actual amount of gDNA present in the sample, where the target DNA was present in the sample. We estimated the amount of mRNA at each probe using this model, and scaled the amounts to the median intensity across the kanMX gene to make mRNA levels comparable across constructs. All files reference the sequences contained within the accompanying ".seq.txt" files. Note: the coordinates in these files do not correspond to genomic coordinates as the construct was integrated into the genome (thereby altering the length of the chromosome). However, positions 1..5001 in these files corresponds to positions 42000..47000 of chromosome 4 in the yeast genome (May, 2008 genome version) and positions 12341..20888 correspond to positions 47800..56347 of chromosome 4.
2013-09-01 | E-GEOD-47004 | biostudies-arrayexpress
Project description:This experiment aims to map nucleosome positions and comparison of the same in WT NORMAL GROWTH vs WT-NUTRIENT STARVATION/isw1∆2∆ MUTANT/rsc4-∆4 MUTANT in Saccharomyces cerevisiae using a custom designed tiling array on Agilent plat form. The corresponding platform is submitted to GEO under Geo-ID GPL15842. 60mer probes with variable tiling density were designed for all the genes transcribed by RNA polymerase III. Each gene is tiled along with its 1kb downstream and upstream region with the exceptions of RPR1, SCR1, RDN5(1-6) and SNR52. Mononucleosomal DNA and size matched naked DNA was competitively hybridized to the array. Data was extracted and normalized log ratios were calculated using Agilent sofware. Normalized log2 ratio data was used in MLM to detection nucleosome positions.
2013-06-21 | GSE39802 | GEO
Project description:ChIP-chip on custom NimbleGen genomic tiling arrays
| PRJNA105111 | ENA
Project description:Using criteria based on known examples, we identified approximately 1.7 million and 2.4 million sequences encoding putative miRNA precursor (stem-loop) structures in the mouse and human genomes, respectively, as well as large numbers of such structures in the genomes of fish, fruitfly and nematode worm. These predictions were then tested using high density custom microarrays containing modified oligonucleotides interrogated with purified small RNAs from different tissues. We found that 19% of 4,006 randomly chosen mouse predictions and 40% of 1,859 randomly chosen human predictions gave positive signals with small RNA isolated from whole 10-12 day embryos and a mixture of brain and testis, respectively, 84% of which hybridized to only one strand of the predicted stem-loop structure, whereas only 2% and 12% of equivalent random mouse and human predictions were positive. There was no difference in the validation rates between sequences exhibiting conservation and those that did not. High validation rates were also observed with predictions from fish, fly and worm genomes. Northern blot analysis of the array-positive mouse predictions confirmed that the vast majority of these sequences were detectable as small RNAs whose sizes ranged from 20-110 nucleotides. Taking into account false negative rates of the prediction and detection of known miRNAs, and assuming that this holds for other small RNAs, we estimate that there are 1 million small RNAs expressed in mouse and 3 million small RNAs in human. Keywords: genomic survey of small RNAs
2009-12-07 | GSE7217 | GEO
Project description:STAT1 ChIP-chip performed on Human Hela S3 Cells for three different platforms. Nimblegen ENCODE arrays which comprise 50mer oligonucleotides spaces every 38bps (overlapping by 12nts) (5 biological replicates), custom maskless array tiling most of ENCODE with 50mer oligonucleotides end-to-end (3 biological replicates) and custom maskless array tiling most of ENCODE with 36mer oligonucleotides end-to-end (2 biological replicates). The chromatin-immunoprecipitation protocol is the same for all samples, however the labelling and hybridization protocols differ between Nimblegen and custom maskless arrays. Keywords = Transcription Factor Binding, STAT1, ChIP-chip, Human, Genome Tiling Arrays Keywords: other
2005-05-27 | GSE2714 | GEO
Project description:The aim of this study is to discover loss of specific miRNA loci in Wilms' tumors using array CGH. Custom arrays were designed based on the Agilent 2x105K Human Whole Genome Genomic microarray with Agilent’s eArray program (https://earray.chem.agilent.com/earray/), with additional probes that cover all miRNA regions (200 bps before, within and after each miRNA from miRBase v13, with each probe in triplicates to enhance the reliability). All custom-designed probes were designed in UCSC hg18. Probes from Agilent’s database were lifted-over from hg19 to hg18 (LiftOver tool: http://genome.ucsc.edu).
2012-03-31 | GSE28397 | GEO
Project description:Gene expression variation was measured in 17 non-laboratory strains compared to the sequenced S288c lab strain Keywords: comparative genomic hybridizations (CGH) comparing different yeast strains Each strain was grown in at least biological triplicate to log phase in rich (YPD) medium. Extracted total RNA was compared to that collected from the diploid S288C strain, DBY8268 (ura3-52/ura3-delta, ho/ho, GAL2/GAL2)
2010-06-19 | E-GEOD-10267 | biostudies-arrayexpress
Project description:We designed a custom expression 8x15 k microarray for Cottus fishes based on transcriptome sequencing. It is a known fact, that oligonucleotide probes differ in the binding behavior towards their target sequences. Therefore, we performed a calibration of our microarray where we assessed the binding behavior of the individual probes empirically. This information was used to normalize gene expression data measurements with the same microarray in another experiment. Please refer to the accompanying publication (Czypionka et al. 2011."Transcriptome changes after genome wide admixture in invasive sculpins" Molecular Ecology; no doi yet) for more information.
2012-03-27 | GSE36800 | GEO
Project description:Gene expression variation was measured in 17 non-laboratory strains compared to the sequenced S288c lab strain Keywords: Gene expression comparisons in different yeast strains Each strain was grown in at least biological triplicate to log phase in rich (YPD) medium. Extracted total RNA was compared to that collected from the diploid S288C strain, DBY8268 (ura3-52/ura3-delta, ho/ho, GAL2/GAL2)
2010-06-19 | E-GEOD-10268 | biostudies-arrayexpress
Project description:A library of transposon mutants was generated in Salmonella enterica serovar Typhimurium strain SL1344 using custom Tn5 and Mu transposons with outward-facing T7 and SP6 promoters. The library was grown up in vitro, and used as the input pool for mouse infection experiments. An in vivo output pool was obtained from the mouse livers 2 days post-infection.<br><br>Genomic DNA was extracted from the input and output pools and digested with the restriction enzyme RsaI. Cy5-labelled RNA run-offs were produced seperately from the T7 and SP6 promoters and hybridised to a tiling microarray along with the Cy3-labelled in vitro transcription products from an untransposed SL1344 wild type control. Analysis of the microarray data allows the location of the transposon inserts to be determined. Mutants that are present in the input pool but that are absent or less prevalent in the output pools are inferred to be attenuated in vivo.
2009-08-25 | E-MEXP-2076 | biostudies-arrayexpress