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).
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