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Sample GSM1252505 Query DataSets for GSM1252505
Status Public on Oct 31, 2013
Title ctrl_rep2
Sample type RNA
 
Channel 1
Source name whi3-dRRM-TAP
Organism Saccharomyces cerevisiae
Characteristics strain background: W303
genotype/variation: whi3-dRRM-TAP
sample type: immunoprecipated RNA
Growth protocol Cells were grown in YPD at 30°C to log phase for harvest.
Extracted molecule total RNA
Extraction protocol To immunoprecipitate RNA, 8L of yeast cells carrying Whi3 or Whi3-dRRM tagged with the TAP tag were grown in YP to log phase. Cells were harvested, resuspended in cold lysis buffer (10mM Hepes-Na, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT and protease inhibitors) and lysed by passing through a French press twice. KCl and Triton were added to the extract to a final concentration of 200 mM KCl and 1% Triton. After centrifugation at 4000 rpm for 5 min and 15000 rpm for 15 min at 4°C, aliquots of the supernatants were saved for assays of input protein and RNA, and the rest was passed through columns filled with 100 µl IgG Sepharose 6 Fast Flow (GE Healthcare) (The protein A moiety of the TAP tag binds to IgG.). After three washes with IPP150 (10mM Tris-Cl, pH 8, 150 mM NaCl, 0.1% Triton), the IgG beads were resuspended in 600 µl TES (10mM Tris-HCl, pH8, 10mM EDTA, pH8, 0.5% SDS). To isolate the RNA associated with beads, equal volume of acid phenol was added, vortexed and incubated at 65°C for 15 min. After two rounds of acid phenol extraction, the isolated RNA was ethanol precipitated from the aqueous phase for labeling. Total RNA used for the microarray referece was extracted by acid phenol method.
Label Cy3
Label protocol 20-25 µg input RNA and all of the immunoprecipitated RNA was used for labeling by an aminoallyl labeling method adapted from The Institute for Genomic Research, Standard Operating Procedure SOP #M004. Briefly, the RNA was reverse transcribed with SuperScript®II (invitrogen) and Oligo-dT, 2mM dNTPs and 0. 3mM amino-allyl-dUTP (Ambion) was added for incorporation into the cDNA. The labeled cDNA was purified and coupled with the appropriate NHS-ester Cy-dye (Amersham). All the labeled cDNAs from immunoprecipitated RNA were hybridized against total RNA containing 50 pmol of incorporated dye.
 
Channel 2
Source name whi3-dRRM-TAP
Organism Saccharomyces cerevisiae
Characteristics strain background: W303
genotype/variation: whi3-dRRM-TAP
sample type: reference RNA
Growth protocol Cells were grown in YPD at 30°C to log phase for harvest.
Extracted molecule total RNA
Extraction protocol To immunoprecipitate RNA, 8L of yeast cells carrying Whi3 or Whi3-dRRM tagged with the TAP tag were grown in YP to log phase. Cells were harvested, resuspended in cold lysis buffer (10mM Hepes-Na, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT and protease inhibitors) and lysed by passing through a French press twice. KCl and Triton were added to the extract to a final concentration of 200 mM KCl and 1% Triton. After centrifugation at 4000 rpm for 5 min and 15000 rpm for 15 min at 4°C, aliquots of the supernatants were saved for assays of input protein and RNA, and the rest was passed through columns filled with 100 µl IgG Sepharose 6 Fast Flow (GE Healthcare) (The protein A moiety of the TAP tag binds to IgG.). After three washes with IPP150 (10mM Tris-Cl, pH 8, 150 mM NaCl, 0.1% Triton), the IgG beads were resuspended in 600 µl TES (10mM Tris-HCl, pH8, 10mM EDTA, pH8, 0.5% SDS). To isolate the RNA associated with beads, equal volume of acid phenol was added, vortexed and incubated at 65°C for 15 min. After two rounds of acid phenol extraction, the isolated RNA was ethanol precipitated from the aqueous phase for labeling. Total RNA used for the microarray referece was extracted by acid phenol method.
Label Cy5
Label protocol 20-25 µg input RNA and all of the immunoprecipitated RNA was used for labeling by an aminoallyl labeling method adapted from The Institute for Genomic Research, Standard Operating Procedure SOP #M004. Briefly, the RNA was reverse transcribed with SuperScript®II (invitrogen) and Oligo-dT, 2mM dNTPs and 0. 3mM amino-allyl-dUTP (Ambion) was added for incorporation into the cDNA. The labeled cDNA was purified and coupled with the appropriate NHS-ester Cy-dye (Amersham). All the labeled cDNAs from immunoprecipitated RNA were hybridized against total RNA containing 50 pmol of incorporated dye.
 
 
Hybridization protocol Labeled samples were boiled in hybridization buffer ( 25% formamide, 5xSSC, %0.1 SDS, 100ug/mL ssDNA) following resuspension, Boiled sample is then hybridized to the homemade Stonybrook_Redgreengene.com-S.cerevisiae-9.2k-A11 array at 50°C for 16-20 hours. After hybridization, array were rinsed 2x in wash buffer (2xSSC, 1% SDS, 50°C), washed 10 minutes x2 in wash buffer and then rinsed 4x with 1xSSC (RT). Arrays were dried by centrifugation for immediate scan.
Scan protocol Array were scanned on GenePix 4000B (Axon Instrument) controlled by GenePix Pro 5.1 software. Images were quantified using GenePix Pro.
Description Biological replicate 2 of 2. RNA immunoprecipitated by whi3-dRRM
Data processing The net intensity of each spot was calculated by subtracting the median of the local background from the foreground, and the red/green ratio was loess normalized. Flagged spots were removed for analysis. Spot values were averaged for multiple independent spots with the same probe.
 
Submission date Oct 28, 2013
Last update date Oct 31, 2013
Contact name Ying Cai
E-mail(s) cai.ying@stonybrook.edu
Organization name Stony Brook University
Department Molecular Genetics and Microbiology
Lab Bruce Futcher
Street address Life Sciences Building Rm 363
City Stony Brook
State/province New York
ZIP/Postal code 11794
Country USA
 
Platform ID GPL17846
Series (2)
GSE51166 WHI3
GSE51784 Whi3 RIP-Chip

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (Cy3/Cy5) representing immunoprecipitated RNA/poly(A) tailed RNA

Data table
ID_REF VALUE
YHR055C -0.605
YOL138C
YPR161C 1.07325
YDR395W -0.18025
YGR129W 1.347666667
YLL039C-3UTR 2.209
YPR165W -0.706
YPR098C 1.852
YPL015C 0.2585
YCL050C -1.0915
YAL069W -0.2975
YLR031W 1.611
YIL014C-A 1.372
YMR193W 0.494
YGR053C 2.874
YER087C-B -0.7895
YOR280C 0.7325
YGR097W -0.03675
YHR215W 0.259777778
YKL025C 0.5295

Total number of rows: 5870

Table truncated, full table size 91 Kbytes.




Supplementary file Size Download File type/resource
GSM1252505_1303.txt.gz 7.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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