Yeast HTP RNA isolation for robot v2.0: Growth of deletion mutant is done using the 24-well Tecan Plate Reader and subsequent RNA extraction and cleanup for robotic amplification: 1. Take frozen cell pellets from 1.5 ml cultures(-80C) and resuspend in 500 ul Acid Phenol Chloroform (From Sigma, 5:1, pH 4.7). Immediately add the same volume TES-buffer (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). Vortex very hard for 20 seconds to resuspend pellet. 2. Incubate in water bath for 10 minutes at 65C and vortex again. 3. Incubate tubes in the thermomixer (65C, 1400RPM) for 50 minutes. 4. Spin for 20 minutes at 14000 rpm at 4C. 5. Take new Eppendorf-tube, fill with 500 ul Phenol Chloroform (5:1, pH 4.7). Add water-phase from point 3. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C. 6. Take new Eppendorf-tube, fill with 500 ul Chloroform:Isoamyl-alcohol (25:1). Add water-phase from point 4. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C. 7. Take new Eppendorf-tube, fill with 50 ul Sodium Acetate (NaAc 3M, pH 5.2). Add water-phase from point 5. Fill tube with Ethanol (100%, -20C). Flip tubes three times. Incubate at -20C for longer than 30 minutes. 8. Spin tubes down for 5 minutes at room temperature, 14000 rpm. Remove fluid with pipette. Be careful not to touch RNA-pellet. 9. Wash pellet with Ethanol 500 ul (80%, -20C). Spin down shortly. 10. Remove Ethanol, do not air dry but directly proceed to 11. 11. Dissolve RNA-pellet in 93 ul sterile water (MQ, freshly tapped). Pipet until the pellet is released from the side of the tube. Wait 5 minutes and pipet up and down 5 more times. Yield will be around 150ug. (Make sure all RNA is dissolved completely!) 12. RNA solution is transferred to a 96 wells plate (4titude, Bioke). Subsequent protocols are processed automatically on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrophotometer (Molecular Devices), and a magnetic bead-locator (Beckman). RNA cleanup protocol description: * concentration of all RNAs is measured by diluting 5 ul RNA on the SpectraMax; * 12.5 ul DNaseI dilution (RNase-free DNase kit, Qiagen, nr 79254, diluted 1:5 in RDD)is mixed with remaining 87.5 ul RNA-solution and incubated @ 18C for 15 minutes; * RNA is purified and concentrated with RNAClean (Agencourt, Beckman) according to manufacturers protocol, to an end volume of 25 ul; * 4 ul cleaned RNA is diluted and measured on the SpectraMAX, concentrations are normalized to 0.2 ug/ul in each well; * 5 ul of cleaned and normalized RNA is used to set up a startplate for RNA amplification. * 1 ul of cleaned and normalized RNA is used to check integrity by running on a QiaXcel system. * All plates are snap frozen and stored at -80C until further use.
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 spectrophotometer (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 script. * 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. * 106 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 manufacturers 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 manufacturers protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snap frozen 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 manufacturers 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.
Yeast HTP RNA isolation for robot v2.0: Growth of deletion mutant is done using the 24-well Tecan Plate Reader and subsequent RNA extraction and cleanup for robotic amplification: 1. Take frozen cell pellets from 1.5 ml cultures(-80C) and resuspend in 500 ul Acid Phenol Chloroform (From Sigma, 5:1, pH 4.7). Immediately add the same volume TES-buffer (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). Vortex very hard for 20 seconds to resuspend pellet. 2. Incubate in water bath for 10 minutes at 65C and vortex again. 3. Incubate tubes in the thermomixer (65C, 1400RPM) for 50 minutes. 4. Spin for 20 minutes at 14000 rpm at 4C. 5. Take new Eppendorf-tube, fill with 500 ul Phenol Chloroform (5:1, pH 4.7). Add water-phase from point 3. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C. 6. Take new Eppendorf-tube, fill with 500 ul Chloroform:Isoamyl-alcohol (25:1). Add water-phase from point 4. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C. 7. Take new Eppendorf-tube, fill with 50 ul Sodium Acetate (NaAc 3M, pH 5.2). Add water-phase from point 5. Fill tube with Ethanol (100%, -20C). Flip tubes three times. Incubate at -20C for longer than 30 minutes. 8. Spin tubes down for 5 minutes at room temperature, 14000 rpm. Remove fluid with pipette. Be careful not to touch RNA-pellet. 9. Wash pellet with Ethanol 500 ul (80%, -20C). Spin down shortly. 10. Remove Ethanol, do not air dry but directly proceed to 11. 11. Dissolve RNA-pellet in 93 ul sterile water (MQ, freshly tapped). Pipet until the pellet is released from the side of the tube. Wait 5 minutes and pipet up and down 5 more times. Yield will be around 150ug. (Make sure all RNA is dissolved completely!) 12. RNA solution is transferred to a 96 wells plate (4titude, Bioke). Subsequent protocols are processed automatically on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrophotometer (Molecular Devices), and a magnetic bead-locator (Beckman). RNA cleanup protocol description: * concentration of all RNAs is measured by diluting 5 ul RNA on the SpectraMax; * 12.5 ul DNaseI dilution (RNase-free DNase kit, Qiagen, nr 79254, diluted 1:5 in RDD)is mixed with remaining 87.5 ul RNA-solution and incubated @ 18C for 15 minutes; * RNA is purified and concentrated with RNAClean (Agencourt, Beckman) according to manufacturers protocol, to an end volume of 25 ul; * 4 ul cleaned RNA is diluted and measured on the SpectraMAX, concentrations are normalized to 0.2 ug/ul in each well; * 5 ul of cleaned and normalized RNA is used to set up a startplate for RNA amplification. * 1 ul of cleaned and normalized RNA is used to check integrity by running on a QiaXcel system. * All plates are snap frozen and stored at -80C until further use.
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 spectrophotometer (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 script. * 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. * 106 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 manufacturers 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 manufacturers protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snap frozen 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 manufacturers 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 wild-type hybridizations, as described in Margaritis, Lijnzaad et al., Mol. Sys. Biol. 2009.
