growth_aa_labelling: Cells were grown for two doublings in synthetic complete (SC) medium containing 2% glucose. Rapamycin was added for the indicated amount of time at a final concentration of 7.5 uM (standard) or 0.15 uM (slow depletion). 4-thiouracil (4tU) was added to a final concentration of 5 mM six minutes before harvesting by spinning at 3952 x g for 1 minute. The supernatant was decanted and the cells were snap-frozen in liquid nitrogen.
Extracted molecule
polyA RNA
Extraction protocol
Labelled 4thiouracil fraction: Separation of labeled RNA : 1) Labeling reaction; (use 100 µg total RNA); Heat RNA samples for 10 min at 60 degC, put on ice for 2 min; RNase free 10 x biotinylation buffer (10 mL: 100 mM Tris, pH 7.5, 10 mM EDTA, RNase_free H2O, Store in aliquots of 1 mL at 4 degC). Biotin-HPDP: Stock concentration: 1 mg/ml dissolved in dimethylformamide ( Stock can be aliquoted and stored frozen). For 100 µg RNA: 200 µl biotin-HDPD, 100 µl 10 x biotinylation buffer, 600 µl RNase-free H2O . Incubate at RT for 1.5hr 800 rpm in Eppendorf mixer at 24oC. Spin empty phase lock tubes 1 min at 16,000 rpm before use . Add RNA, Add app. equal volume chloroform to tubes (fill tubes), Mix vigorously; Spin at 20,000 rpm for 5 min at 4 degC. Transfer upper phase into new 2 ml tubes. 2) RNA precipitation: Add 1/10 vol 5M NaCl, mix; Add equal vol of isopropanol (app. 1 ml), mix and spin at 20,000 rpm for 30 min at 4oC. Remove supernatant; Add 1 ml of 75 % ethanol; Spin at 20,000 rpm for 10 min at 4oC; Remove supernatant, quickspin and remove rest of supernatant (don’t let RNA dry). Resuspend RNA in 100 µL RNase-free H20; Measure RNA concentration. 3) Separation of labeled and unlabeled RNA: Washing buffer (100 mM Tris, pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1 % Tween 20, RNase-free H2O).Heat washing buffer to 65oC. Elution Buffer (from - 20 oC aliquots or prepare fresh: 100 mM dithiothreitol (MW 154.25) in RNase-free H2O, need 200 µl / rxn). Heat biotinylated RNA to 65 degC for 10 min and immediately place on ice for 5 min; Add 100 µL of streptavidin beads to biotinylated RNA (max. 100 µl), final app 200 µl; Incubate with slight shaking for 15 min at RT/ 24 oC; Place µMacs columns into magnetic stand. Add 900 µl of RT washing buffer to columns (pre-run and equilibrate); (to initiate the flow through the column you can gently press on the top of the column with your finger); Apply beads/RNA mix (200 µl) to the columns. Labeled RNA fraction: Collect FT in 1.5 ml tubes and apply to column again; 1st wash (400 µL of 65oC washing buffer); Wash 5 x with 600, 700, 800, 900, 1000 µl washing buffer . Pipet 700 µl RLT buffer (RNeasy MinElute Cleanup Kit) into new tubes; Elute RNA directly into RLT buffer with 100 µl elution buffer; Perform a second round of elution (100 µl) into the same tubes after 5 min (final vol 900 µl). Continue with the RNeasy MinElute Cleanup protocol: Add 500 µL 96-100 % ethanol to the diluted RNA and mix thoroughly by pipetting; Do not centrifuge; Apply 700 µl of the sample to an RNeasy MinElute spin column in a 2 ml collection tube; Close the tube gently and spin for 15 s at > 8,000 g; Discard the FT; Apply remaining sample and repeat spin, discard FT; Transfer the spin column to new 2 ml collection tube; Pipet 500 µL RPE buffer onto the spin column, close the tube gently and spin 15 s at > 8,000 g; Discard the FT; Add 500 µL of 80 % ethanol to the spin column, close the tube gently and spin at > 8,000 g, discard the FT and collection tube; Transfer the spin column to a new 2 mL collection tube, open the cap of the spin column and spin at full speed for 5 min, discard FT and collection tube; To elute, transfer the spin column to a new 1.5 ml collection tube; Pipet 20 µl RNase-free H2O directly onto the center of the membrane; Close the tube gently and spin for 1 min at maximum speed; Measure RNA concentration.
Label
Cy5
Label protocol
Robot amplification and labeling v1.0: All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman). mRNA amplification protocol description: * All RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot scriptP * Mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well. * Plate is incubated @ 70C for 10 mins, cooled to 48C. * Mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and 1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well. * Plate is incubated @ 48C for 2 hours and cooled to room temperature. * 10^6 ul water and subsequently mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNaseH (Promega) is added and mixed in each well. * Plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturer's protocol, to an end volume of 25 ul . * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems). * Plate is incubated @37C for 4 hours. * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturer's protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C. Labeling protocol description: * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10). All subsequent steps are performed by the robot script: * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines Cy3 and Cy5 labeled material). * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells. * 3 ul Cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour. * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes. * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturer's protocol. * RNA concentration and labeling incorporation are measured (SpectraMax 190). * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C. * Samples are stored at -20C until hybridization.
growth_aa_labelling: Cells were grown for two doublings in synthetic complete (SC) medium containing 2% glucose. Rapamycin was added for the indicated amount of time at a final concentration of 7.5 uM (standard) or 0.15 uM (slow depletion). 4-thiouracil (4tU) was added to a final concentration of 5 mM six minutes before harvesting by spinning at 3952 x g for 1 minute. The supernatant was decanted and the cells were snap-frozen in liquid nitrogen.
Extracted molecule
polyA RNA
Extraction protocol
Labelled 4thiouracil fraction: Separation of labeled RNA : 1) Labeling reaction; (use 100 µg total RNA); Heat RNA samples for 10 min at 60 degC, put on ice for 2 min; RNase free 10 x biotinylation buffer (10 mL: 100 mM Tris, pH 7.5, 10 mM EDTA, RNase_free H2O, Store in aliquots of 1 mL at 4 degC). Biotin-HPDP: Stock concentration: 1 mg/ml dissolved in dimethylformamide ( Stock can be aliquoted and stored frozen). For 100 µg RNA: 200 µl biotin-HDPD, 100 µl 10 x biotinylation buffer, 600 µl RNase-free H2O . Incubate at RT for 1.5hr 800 rpm in Eppendorf mixer at 24oC. Spin empty phase lock tubes 1 min at 16,000 rpm before use . Add RNA, Add app. equal volume chloroform to tubes (fill tubes), Mix vigorously; Spin at 20,000 rpm for 5 min at 4 degC. Transfer upper phase into new 2 ml tubes. 2) RNA precipitation: Add 1/10 vol 5M NaCl, mix; Add equal vol of isopropanol (app. 1 ml), mix and spin at 20,000 rpm for 30 min at 4oC. Remove supernatant; Add 1 ml of 75 % ethanol; Spin at 20,000 rpm for 10 min at 4oC; Remove supernatant, quickspin and remove rest of supernatant (don’t let RNA dry). Resuspend RNA in 100 µL RNase-free H20; Measure RNA concentration. 3) Separation of labeled and unlabeled RNA: Washing buffer (100 mM Tris, pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1 % Tween 20, RNase-free H2O).Heat washing buffer to 65oC. Elution Buffer (from - 20 oC aliquots or prepare fresh: 100 mM dithiothreitol (MW 154.25) in RNase-free H2O, need 200 µl / rxn). Heat biotinylated RNA to 65 degC for 10 min and immediately place on ice for 5 min; Add 100 µL of streptavidin beads to biotinylated RNA (max. 100 µl), final app 200 µl; Incubate with slight shaking for 15 min at RT/ 24 oC; Place µMacs columns into magnetic stand. Add 900 µl of RT washing buffer to columns (pre-run and equilibrate); (to initiate the flow through the column you can gently press on the top of the column with your finger); Apply beads/RNA mix (200 µl) to the columns. Labeled RNA fraction: Collect FT in 1.5 ml tubes and apply to column again; 1st wash (400 µL of 65oC washing buffer); Wash 5 x with 600, 700, 800, 900, 1000 µl washing buffer . Pipet 700 µl RLT buffer (RNeasy MinElute Cleanup Kit) into new tubes; Elute RNA directly into RLT buffer with 100 µl elution buffer; Perform a second round of elution (100 µl) into the same tubes after 5 min (final vol 900 µl). Continue with the RNeasy MinElute Cleanup protocol: Add 500 µL 96-100 % ethanol to the diluted RNA and mix thoroughly by pipetting; Do not centrifuge; Apply 700 µl of the sample to an RNeasy MinElute spin column in a 2 ml collection tube; Close the tube gently and spin for 15 s at > 8,000 g; Discard the FT; Apply remaining sample and repeat spin, discard FT; Transfer the spin column to new 2 ml collection tube; Pipet 500 µL RPE buffer onto the spin column, close the tube gently and spin 15 s at > 8,000 g; Discard the FT; Add 500 µL of 80 % ethanol to the spin column, close the tube gently and spin at > 8,000 g, discard the FT and collection tube; Transfer the spin column to a new 2 mL collection tube, open the cap of the spin column and spin at full speed for 5 min, discard FT and collection tube; To elute, transfer the spin column to a new 1.5 ml collection tube; Pipet 20 µl RNase-free H2O directly onto the center of the membrane; Close the tube gently and spin for 1 min at maximum speed; Measure RNA concentration.
Label
Cy3
Label protocol
Robot amplification and labeling v1.0: All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman). mRNA amplification protocol description: * All RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot scriptP * Mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well. * Plate is incubated @ 70C for 10 mins, cooled to 48C. * Mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and 1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well. * Plate is incubated @ 48C for 2 hours and cooled to room temperature. * 10^6 ul water and subsequently mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNaseH (Promega) is added and mixed in each well. * Plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturer's protocol, to an end volume of 25 ul . * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems). * Plate is incubated @37C for 4 hours. * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturer's protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C. Labeling protocol description: * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10). All subsequent steps are performed by the robot script: * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines Cy3 and Cy5 labeled material). * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells. * 3 ul Cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour. * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes. * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturer's protocol. * RNA concentration and labeling incorporation are measured (SpectraMax 190). * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C. * Samples are stored at -20C until hybridization.
Hybridization protocol
Tecan HS4800 hybridization: * 60 ul labeled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA * Hybridizations of spotted oligo-arrays (Codelink glass) or Agilent microarrays are performed on a HS4800Pro Hybstation (Tecan) * Priming: 5xSSC, 0.1%SDS * Probe injection: pre-hyb, 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, Volume 110ul * Hybridization: 45 min at 42C. * Wash 2x: milliQ * Wash: 5xSSC, 0.1%SDS * Probe injection: sample. Volume 110ul (Agilent 4packs: 55ul) * Hybridization: 16 hours at 42C. * Wash 2x: 1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC at 23C * Drying: blow with nitrogen for 3min at 30C
Scan protocol
Scanning of slides using the Agilent G2565BA scanner. Features were extracted using ImaGene software from Biodiscovery.
Data processing
Genes, no bg corr, dye corr: Normalization was done using print-tip LOESS as described in (Yang et al. 2002), by estimating the LOESS curve for all gene probes using no background subtraction and a span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve. Signals were corrected for dye bias using intrinsic gene-specific dye biases estimated from a set of wild-type hybridizations, as described in Margaritis, Lijnzaad et al., Mol. Sys. Biol. 2009.
