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Sample GSM1276533 Query DataSets for GSM1276533
Status Public on Dec 04, 2013
Title Npl3 Replicate 1
Sample type RNA
 
Channel 1
Source name HKY820
Organism Saccharomyces cerevisiae
Characteristics probe: lysate
co-ip: none
Growth protocol Yeast cells were grown at 25°C in selective medium to a density of 4 x 10^7 cells/ml.
Extracted molecule total RNA
Extraction protocol Yeast cells were grown at 25°C in selective medium to a density of 4 x 107 cells/ml. The cells were collected, washed with 1 x TBS and resuspended in RNA-IP buffer (25 mM Tris-HCl, pH 7.5, 100 mM KCl, 0,2% (v/v) Triton X-100, 0,2 mM PMSF, 5 mM DTT, 10 units RiboLockTM RNase Inhibitor (Thermo Scientific)). One cell pellet volume of glass beads, 1,5 volumes of RNA-IP buffer supplemented with protease inhibitor cocktails (Sigma and Roche) were added. Cells were lysed by vigorous vortexing for 25 sec and 4m/s using the FastPrep®-24 instrument (MP Biomedicals). For DNaseI digestion 800 µl lysate was incubated with 15 µl (40 Kunitz units) DNaseI (Qiagen) at 30°C for 30 min. The co-immunoprecipitation experiments were performed at 4°C for 3 h by incubating lysates with 20 µl of Protein G sepharose beads (Amersham Biosciences) conjugated to monoclonal c-myc (9E10)-antibody (Santa Cruz) or GFP-Trap®-beads (ChromoTek). The beads were washed five times with RNA-IP buffer and the RNA was eluted in 100 µl DEPC-treated ddH2O during incubation at 65°C for 10 min. The RNA eluate was purified via phenol-chloroform extraction followed by an ethanol precipitation. The RNA was finally eluted in 10 µl of DEPC-treated ddH2O. 100 µl of the lysate was collected as an input control before co-immunoprecipitation and wasand was purified via phenol-chloroform extraction followed by an ethanol precipitation and eluted in 10-20 µl in parallel to the eluate (co-immunoprecipitated RNA) to obtain a total input sample for Microarray analyses. RNA co-immunoprecipitations were performed in two independent experiments each for myc-tagged Gbp2, Hrb1 and Npl3.
Label Cy3
Label protocol The RNA probes from RNA Co-immunoprecipitations were qualitatively analyzed by ExperionTM (Biorad). One microgram of RNA from lysate was amplified using the Message AmpTM II aRNA Amplification kit (Ambion). 4 μg of an RNA sample was transcribed into Cy5 labeled cDNA using the AmershamTM CyScribeTM cDNA Post-labelling kit (GE Healthcare). Unincorporated nucleotides were removed following the manufacturer's protocol of illustraTM CyScribeTMGFXTM Purification kit (GE Healthcare).
 
Channel 2
Source name HKY820
Organism Saccharomyces cerevisiae
Characteristics probe: eluate
co-ip: Npl3
Growth protocol Yeast cells were grown at 25°C in selective medium to a density of 4 x 10^7 cells/ml.
Extracted molecule total RNA
Extraction protocol Yeast cells were grown at 25°C in selective medium to a density of 4 x 107 cells/ml. The cells were collected, washed with 1 x TBS and resuspended in RNA-IP buffer (25 mM Tris-HCl, pH 7.5, 100 mM KCl, 0,2% (v/v) Triton X-100, 0,2 mM PMSF, 5 mM DTT, 10 units RiboLockTM RNase Inhibitor (Thermo Scientific)). One cell pellet volume of glass beads, 1,5 volumes of RNA-IP buffer supplemented with protease inhibitor cocktails (Sigma and Roche) were added. Cells were lysed by vigorous vortexing for 25 sec and 4m/s using the FastPrep®-24 instrument (MP Biomedicals). For DNaseI digestion 800 µl lysate was incubated with 15 µl (40 Kunitz units) DNaseI (Qiagen) at 30°C for 30 min. The co-immunoprecipitation experiments were performed at 4°C for 3 h by incubating lysates with 20 µl of Protein G sepharose beads (Amersham Biosciences) conjugated to monoclonal c-myc (9E10)-antibody (Santa Cruz) or GFP-Trap®-beads (ChromoTek). The beads were washed five times with RNA-IP buffer and the RNA was eluted in 100 µl DEPC-treated ddH2O during incubation at 65°C for 10 min. The RNA eluate was purified via phenol-chloroform extraction followed by an ethanol precipitation. The RNA was finally eluted in 10 µl of DEPC-treated ddH2O. 100 µl of the lysate was collected as an input control before co-immunoprecipitation and wasand was purified via phenol-chloroform extraction followed by an ethanol precipitation and eluted in 10-20 µl in parallel to the eluate (co-immunoprecipitated RNA) to obtain a total input sample for Microarray analyses. RNA co-immunoprecipitations were performed in two independent experiments each for myc-tagged Gbp2, Hrb1 and Npl3.
Label Cy5
Label protocol The RNA probes from RNA Co-immunoprecipitations were qualitatively analyzed by ExperionTM (Biorad). One microgram of RNA from lysate was amplified using the Message AmpTM II aRNA Amplification kit (Ambion). 4 μg of an RNA sample was transcribed into Cy5 labeled cDNA using the AmershamTM CyScribeTM cDNA Post-labelling kit (GE Healthcare). Unincorporated nucleotides were removed following the manufacturer's protocol of illustraTM CyScribeTMGFXTM Purification kit (GE Healthcare).
 
 
Hybridization protocol Yeast microarrays (Y6.4k6; University Health Network Microarray Centre, Toronto, Canada) were pre-hybridized at 55°C for 30 min in 5 x SSC, 0.1 % SDS and 1 % bovine serum albumin and rinsed 5 times with water. The slides were dried by spinning them for 3 min at 1500 rpm. Labelled probes were supplemented with 3 μl of yeast tRNA (10 mg/ml) and 3 μl of denatured salmon sperm DNA (10 mg/ml). Probes were concentrated in a speed vac to a final volume of 26 μl and supplemented with 4 μl 2% bovine serum albumin and 10 μl 20 x SSC before they were spotted onto a microarray slide. The hybridization was carried out for 16 h at 55°C in a hybridization chamber. After removal of the HybriSlipTM Hybridization Cover (Sigma-Aldrich) the slides were washed with agitation three times in 1x SSC buffer containing 0.1 % SDS, two times with 1 x SSC buffer and once in ddH2O. Arrays were dried by centrifugation.
Scan protocol Fluorescence intensities were monitored by using the scanner ScanArray Express (PerkinElmer Life Sciences) using ScanArray Express, Microarray Analysis System 4.0.0.4.
Data processing quantile normalization of median signales using the R-package limma
 
Submission date Nov 27, 2013
Last update date Dec 04, 2013
Contact name Klaus Jung
Organization name University Medical Center Göttingen
Department Medical Statistics
Street address Humboldtallee 32
City Göttingen
ZIP/Postal code D-37073
Country Germany
 
Platform ID GPL10165
Series (1)
GSE52808 Quality control of spliced mRNAs requires the shuttling SR-proteins Gbp2 and Hrb1

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (Cy5/Cy3) representing eluate/lysate

Data table
ID_REF VALUE
1 -0.231820794
3 -0.215333163
5 0.043416955
7 -0.182536447
9 0.345161707
11 0.46315181
13 0.016827909
15 -0.039752951
17 -0.251942884
19 -0.514627607
21 0.566265972
23 -0.895473007
25 -0.020680215
27 0.006642115
29 -1.057441084
31 -1.083914575
33 0.58716274
35 -1.156353523
37 -0.197457745
39 0.462691878

Total number of rows: 6295

Table truncated, full table size 107 Kbytes.




Supplementary file Size Download File type/resource
GSM1276533_Npl3_Replicate1.txt.gz 1.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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