1 sample(s) found by the keyword GSM254979.

  1. GEO sample ID: GSM254979
    • Sample_geo_accession: GSM254979
    • Sample_status: Public on Jan 07 2008
    • Sample_submission_date: Jan 07 2008
    • Sample_last_update_date: Jan 07 2008
    • Sample_type: other
    • Sample_channel_count: 1
    • Sample_source_name_ch1: RNA purified from control cells_sub-polysomal fraction of polyribosomes
    • Sample_organism_ch1: Saccharomyces cerevisiae
    • Sample_characteristics_ch1: strain BY4741
    • Sample_treatment_protocol_ch1: 100 mL cultures were grown to OD600nm 0.5, when DTT was added to a final concentration of 2mM (or an equivalent volume of water was added to control cultures), and flasks were returned to the incubator for a further 1 hour. Cyclohexomide was added to a final concentration of 0.1mg/mL, and flasks were cooled immediately on ice.
    • Sample_growth_protocol_ch1: S. cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met5Δ0 ura3Δ0) was grown in YEPD (2% w/v bacto-peptone, 2% w/v glucose, 1% w/v yeast extract) adjusted to pH 5.4, with 200rpm rotary aeration at 30oC.
    • Sample_molecule_ch1: other
    • Sample_extract_protocol_ch1: Cells were spun down (6000rpm, 4mins, 4oC), and washed twice in 2.5 mL lysis buffer (20mM Tris-HCl pH 8.0, 140mM KCl, 1.5mM MgCl2, 0.5mM DTT, 1% v/v Triton X-100, 0.1mg/mL cycloheximide, 1mg/mL heparin). Following resuspension in 0.7 mL lysis buffer, cells were transferred to a chilled 15 mL corex tube, and 0.5 mL chilled glass beads (0.45-0.55mm) were added. Cells were vortexed 4 times for 20 secs each, with 90 secs on ice between. Cells were then spun (5000rpm, 5 mins, 4oC), and the supernatant was transferred to a chilled 1.5 mL tube, and spun again (9500rpm, 5 mins, 4oC). The supernatant was loaded onto an 11 mL 15-60% sucrose gradient, prepared in 20mM Tris-HCl pH 8.0, 140mM KCl, 5mM MgCl2, 0.5mM DTT, 0.1mg/mL cycloheximide, 0.5mg/mL heparin, in a 12 mL ultra-clear tube (Beckman). Sucrose gradients were spun at 35000rpm for 2.5 hours at 4oC. Using a programmable density gradient system (ISCO), 0.5 mL fractions were collected from the sucrose gradients into 1.5 mL tubes containing 1 mL of 6M guanidine HCl, mixed by inversion and immediately chilled on ice. Fractions were pooled into non-polysomal and polysomal samples according to OD254nm profiles. Non-polysomal and polysomal samples from 3 gradients were pooled together. Following addition of an equal volume of ethanol, pools were precipitated overnight at -20oC. RNA was spun down (10000rpm, 20 mins, 4oC), washed in 2 mL 70% ethanol, and resuspended in 400 μL TE buffer. RNA was re-precipitated by addition of 0.1 volume 3M Sodium acetate pH 5.2 and 2 volumes ethanol, at -20oC for 1 hour. RNA was spun down as before, washed in 1 mL 70% ethanol, and resuspended in 0.5 mL water. Any residual protein was removed by extraction with phenol:chloroform (1:1 v/v) and chloroform:IAA (24:1 v/v). An equal volume of 4M LiCL was added to the aqueous phase, and RNA was precipitated overnight at -20oC. RNA was spun down as before, washed in 1 mL 70% ethanol, resuspended in 400 μL water, and re-precipitated with 0.1 volume sodium acetate pH 5.2, 2 volumes ethanol. RNA was washed in 1 mL 70% ethanol, and resuspended in 20 μL water.
    • Sample_label_ch1: biotin
    • Sample_label_protocol_ch1: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 μg total RNA (Affymetrix Manual II).
    • Sample_hyb_protocol: Following fragmentation, 20 μg of cRNA were hybridized for 16 hr at 45oC on high-density oligonucleotide Yeast Genome S98 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
    • Sample_scan_protocol: GeneChips were scanned using the Affymetrix GeneArray 3000 Scanner, according to the Affymetrix Manual II.
    • Sample_description: Polyribosomes following DTT or control treatment
    • Sample_data_processing: The data were analysed with Affymetrix CGOS software. The average intensity of all probe seta was used for normalisation and scaled to 100 in the absolute analysis for each probe array.
    • Sample_platform_id: GPL90
    • Sample_contact_name: Colin,,Hanfrey
    • Sample_contact_email: colin.hanfrey@bbsrc.ac.uk
    • Sample_contact_laboratory: Molecular Metabolism
    • Sample_contact_institute: Institute of Food Research
    • Sample_contact_address: Norwich Research Park
    • Sample_contact_city: Norwich
    • Sample_contact_state: Norfolk
    • Sample_contact_zip/postal_code: NR4 7UA
    • Sample_contact_country: United Kingdom
    • Sample_supplementary_file: ftp://ftp.ncbi.nih.gov/pub/geo/DATA/supplementary/samples/GSM254nnn/GSM254979/GSM254979.CEL.gz
    • Sample_supplementary_file: ftp://ftp.ncbi.nih.gov/pub/geo/DATA/supplementary/samples/GSM254nnn/GSM254979/GSM254979.EXP.gz
    • Sample_series_id: GSE10091
    • Sample_data_row_count: 9335
    • Sample_comment: Raw data provided as supplementary file
    • sample_table_begin:
    • sample_table_end:
    • Sample_title: control cells_polysomal
    ID_REF Corrected Value VALUE ABS_CALL
    AFFX-MurIL2_at 1 4.3 A
    AFFX-MurIL10_at 1.9 2.8 A
    AFFX-MurIL4_at 4.9 0.7 A
    AFFX-MurFAS_at 10.1 2 A
    AFFX-BioB-5_at 78.8 45.9 A
    AFFX-BioB-M_at 118.1 91 P
    AFFX-BioB-3_at 131.4 107.1 P
    AFFX-BioC-5_at 205.3 177.4 P
    AFFX-BioC-3_at 199.5 167 P
    AFFX-BioDn-5_at 175.6 141.8 P
    View Full Table

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