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.
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 1 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.
