1 sample(s) found by the keyword GSM189735.


  1. GEO sample ID: GSM189735
    • Sample_geo_accession: GSM189735
    • Sample_status: Public on Dec 21 2007
    • Sample_submission_date: May 16 2007
    • Sample_last_update_date: Dec 21 2007
    • Sample_type: RNA
    • Sample_channel_count: 1
    • Sample_source_name_ch1: MLY132_SLAM
    • Sample_organism_ch1: Saccharomyces cerevisiae
    • Sample_characteristics_ch1: gpa2 deletion mutant MLY132 transferred to low nitrogen SLAM media
    • Sample_growth_protocol_ch1: Yeast strains, media and growth conditions Diploid strains MLY61 a/alpha (ura3-52/ura3-52 MATa/alpha) and MLY132 a/alpha (delta gpa2::G418/delta gpa2::G418 ura3-52/ura3-52 MATa/alpha) were obtained from J. Heitman, Duke University{Lorenz, 2000 #19; Lorenz, 1997 #33}. Standard YPM media were used as described{Sherman, 1991 #36}. Synthetic low ammonium media consisting of SLAM (0.17% Yeast Nitrogen Base without ammonium sulfate or amino acids, 50 µM ammonium sulfate, 2% maltose) were used when switching to low nitrogen pseudohyphal inducing conditions{Lorenz, 2000 #19; Lorenz, 1997 #33}. Similar to a previously described method{Pan, 2000 #37}, strains were grown to an OD600 of 1.0 in YPM media. Approximately 200 OD600 units were collected by filtering followed by resuspension in 10 mL RNAlater (Ambion) for <30 min, pelleted by centrifugation, flash frozen and stored at –80 °C. Strains were also grown to an OD600 of 1.0 in YPM media and approximately 200 O.D. were collected by filtration and transferred to SLAM media for 2 h at 30 °C prior to collection as above.
    • Sample_molecule_ch1: total RNA
    • Sample_extract_protocol_ch1: Expression profiling analyses RNA Isolation: The RibopureTM-Yeast kit (Ambion) was used to extract total RNA from yeast cells. Briefly, for each RNAlater-treated sample, cells were pelleted in a microtube and any remaining RNAlater removed. Then lysis buffer, 10% SDS, and 25:24:1 phenol:chloroform:isoamyl alcohol were used to resuspend the pellet, and the mixture was transferred to a screw cap microtube with Zirconia beads (BioSpec Products) to disrupt the yeast cells by vortexing. The lysate was centrifuged, and the upper aqueous layer transferred to 15 mL conical tubes with Binding Buffer and 100% ethanol. The mixture was drawn through a filter cartridge to capture the RNA, followed by washing and elution. To remove contaminating DNA, 50 µL of the eluted RNA was treated with 10 µL of 10X DNase I buffer and 4 µL of 8 units µL-1 DNase I. The reaction was mixed, incubated at 37 °C for 30 min, followed by addition of 0.1 volume DNase Inactivation Reagent. After 5 min at room temperature, the mixture was spun through a spin column (Qiagen) to remove the DNase binding beads in the DNase Inactivation Reagent. The quality/quantity of purified yeast total RNA was determined by separating 1 µL from each sample on a Bioanalyzer 2100 (Agilent).
    • Sample_label_ch1: biotin
    • Sample_label_protocol_ch1: Target Preparation and Hybridization to Arrays: Eight µg of RNA were concentrated via ethanol precipitation as previously described{Thach, 2003 #81}. All subsequent steps were as described in the GeneChip Expression Analysis Technical Manual version 701021 Rev. 3 (Affymetrix). Briefly, for each sample, the purified, concentrated RNA was reverse-transcribed using a poly-T primer with a T7 RNA-polymerase promoter sequence into double stranded cDNA followed by linear amplification via in vitro transcription (BioArray High Yield IVT kit, ENZO) to yield an antisense cRNA labeled with biotin. The purified biotinylated cRNA was fragmented by magnesium ions and heat and mixed with the hybridization cocktail that contained biotinylated cRNA controls with known concentrations to monitor the quality of the hybridization procedure.
    • Sample_hyb_protocol: The cocktail was hybridized onto the GeneChip® Yeast Genome S98 (YG_S98) microarray (Affymetrix) which contains probe sets for 6,400 genes of the S. cerevisiae S288C strain identified in the Saccharomyces Genome Database (SGD) as of December 1998 and 600 additional probe sets for putative open reading frames (ORFs) identified by SAGE analysis, mitochondrial proteins, TY proteins, plasmids, and a small number of ORFs for strains other than S288C. Hybridization was carried out for 16 h at 45 °C with rotation at 60 rpm. The microarrays were then washed and stained using the Affymetrix GeneChip Fluidics Station 450 and scanned using the GeneChip Scanner 3000.
    • Sample_scan_protocol: The microarrays were then washed and stained using the Affymetrix GeneChip Fluidics Station 450 and scanned using the GeneChip Scanner 3000. GeneChip® Operating Software 1.2 (GCOS 1.2) (Affymetrix) was used to acquire and process array images, determine the quantity and presence or absence of a transcript using the Microarray Suite 5.0 (MAS5) algorithm, and generated the Report files summarizing the quality of target detection for each microarray.
    • Sample_description: Gpa2p deficient diploid yeast strains grown on low nitrogen starved maltose media
    • Sample_data_processing: Data Acquisition, Integration, and Analysis: GeneChip® Operating Software 1.2 (GCOS 1.2) (Affymetrix) was used to acquire and process array images, determine the quantity and presence or absence of a transcript using the Microarray Suite 5.0 (MAS5) algorithm, and generated the Report files summarizing the quality of target detection for each microarray. We used the RMA (robust multi-chip analysis) transcript quantities because this method improved detection of differentially expressed transcripts compared to the MAS5 algorithm{Irizarry, 2003 #78}. However, MAS5 present and absent calls were still used for the filtering of probe sets. Arraytools 3.3.0 developed by Simon and Lam (http://linus.nci.nih.gov/BRB-ArrayTools.html) was used to calculate RMA (robust multi-chip analysis) transcript quantities, integrate transcript data with experimental information such as yeast mutant strains and growth conditions, and to determine differentially expressed transcripts via ANOVA (analysis of variance) and the random variance model. JMP software (SAS) was used to join various data sets together, perform statistical quality control, and determined relations among variables. For joining transcript and protein data, probe set identifiers were mapped to SwissProt identifiers via NetAffx {Liu, 2003 #79} and subsequently matched and joined to protein identifiers from mass spectrometry data output by Pro Group{Applied-Biosystems, 2006 #75}. DAVID 2.0{Dennis, 2003 #77} was used to convert Affymetrix probe set identifiers and Pro Group SwissProt protein identifiers to yeast open reading frames (ORFs) or Saccharomyces Genome Database (SGD) identifiers for input into the SGD GO Term Finder tool (http://db.yeastgenome.org/cgi-bin/GO/goTermFinder) to group up-regulated or down-regulated transcripts or proteins into biological processes. The yeast ORFs derived from DAVID 2.0 were also used for input into the Retrieve Sequence (http://rsat.ccb.sickkids.ca/) tool to obtain sequences for promoter analysis. Up-regulated or down-regulated transcripts from each class comparison were input into the promoter analysis tool {Tompa, 2005 #82; van Helden, 2000 #83}. Promoter analysis was performed on -800 to -1 sequence before the start codons of genes expressing differential transcripts. Sequential putative oligomer or dyad sequences were searched against known transcription factor consensus binding sequences using the Search Putative Sites (http://rulai.cshl.edu/SCPD/searchputative.html) and the Search Existing Motif tools (http://rulai.cshl.edu/SCPD/searchmotif.html) from The Promoter Database of S. cerevisiae (http://rulai.cshl.edu/SCPD/). Matches consist of 6 to 8 basepair identity in a transcription factor’s known/consensus binding site.
    • Sample_platform_id: GPL90
    • Sample_contact_name: Dzung,,Thach
    • Sample_contact_email: dzung.thach@nrl.navy.mil
    • Sample_contact_department: Center for Biomolecular Science and Engineering
    • Sample_contact_institute: Naval Research Laboratory
    • Sample_contact_address: 4555 Overlook Ave SW
    • Sample_contact_city: Washington
    • Sample_contact_state: DC
    • Sample_contact_zip/postal_code: 20375
    • Sample_contact_country: USA
    • Sample_supplementary_file: ftp://ftp.ncbi.nih.gov/pub/geo/DATA/supplementary/samples/GSM189nnn/GSM189735/GSM189735.CEL.gz
    • Sample_series_id: GSE7820
    • Sample_data_row_count: 9335
    • Sample_comment: Raw data provided as supplementary file
    • sample_table_begin:
    • sample_table_end:
    • Sample_title: 10_MLY132_YPM
    ID_REF Corrected Value VALUE
    10000_at 3.690525532 4.012195587
    10001_at 7.81856823 7.80267477
    10002_i_at 10.25229549 10.237957
    10003_f_at 10.29305077 10.28229237
    10004_at 3.891520262 4.108754635
    10005_at 7.721504211 7.769310474
    10006_at 5.895824432 6.142393589
    10007_at 10.01541996 10.2070694
    10008_at 6.248551846 6.52974844
    10009_at 5.909196377 6.180265427
    View Full Table






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