The microarrays experiments of three biological and one technical replicates were performed in YJR12 (wt) and YJR13 (myo1∆) strains. YJR12 (wild type) and YJR13 (myo1∆) strains were obtained as haploid segregants from a cross between YJR6 (myo1::HIS5 strain) and BY4741 (obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) with continuous shaking at 200 rpm.
Extracted molecule
total RNA
Extraction protocol
Cells were harvesting by centrifugation (1,341g for 5min at 4ºC ). Pelleted yeast cells were washed with ice-cold lysis buffer (50mM Tris-HCl, pH 7.5, 100mM NaCl, 7mM MgCl2, 1mM DTT, 1mM PMSF and 50µg/ml cicloheximide) and then resuspended in 1.5ml of lysis buffer together with a quarter volume of acid-washed 0.5mm glass beads. Cells were disrupted by vortex mixing for 20s with 30s intervals on ice (10 times). Unbroken cells and large debris were removed by a low speed spin (800g for 10min). The supernatant was centrifuged at 10,000g for 30 min at 4ºC, yielding supernatant (S10) and pellet (P10). Supernatant S10 was collected and centrifuged at 100,000g in 50% sucrose solution for 60min at 4ºC (Beckman 80 Ti rotor ) thereby yielding supernatant S100 and ribosomal pellet P100. Ribosomal pellets contained in P100 fractions was resuspended in a guanidine thiocyanate buffer containing 10% mercaptoethanol (RLT buffer, RNeasy Mini Kit, Qiagen ). RNA was extracted using the RNeasy Mini Kit for isolation of total RNA (Qiagen, Valencia, CA) following the manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE). The purity and integrity of the RNA was monitored by electrophoresis using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Label
CY3
Label protocol
1.0µg of RNA extracted from ribosomal pellets was amplified using the Low RNA input Fluorescent Linear Amplification kit (Agilent Technologies, Palo Alto, A). Briefly RNA was mixed with 1.2 µL of T7 promoter primer and nuclease free water. The reaction mix was incubated at 65°C for 10 minutes and then reactions was placed on ice for 5 minutes. 8.5 µL of cDNA master mix was added to each reaction and incubated at 40°C for two hours. Then the reaction was incubated at 65°C for 15 minutes. 57.6 µL of transcription master mix including the Cyanine 3-CTP (10mM) was added and then incubated at 40°C for 2 hours, to produce the cRNA. The cRNA was purified using the RNeasy mini spin column from Qiagen and dye incorporation was monitored on an Agilent Bioanalyzer.
The microarrays experiments of three biological and one technical replicates were performed in YJR12 (wt) and YJR13 (myo1∆) strains. YJR12 (wild type) and YJR13 (myo1∆) strains were obtained as haploid segregants from a cross between YJR6 (myo1::HIS5 strain) and BY4741 (obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) with continuous shaking at 200 rpm.
Extracted molecule
total RNA
Extraction protocol
Cells were harvesting by centrifugation (1,341g for 5min at 4ºC ). Pelleted yeast cells were washed with ice-cold lysis buffer (50mM Tris-HCl, pH 7.5, 100mM NaCl, 7mM MgCl2, 1mM DTT, 1mM PMSF and 50µg/ml cicloheximide) and then resuspended in 1.5ml of lysis buffer together with a quarter volume of acid-washed 0.5mm glass beads. Cells were disrupted by vortex mixing for 20s with 30s intervals on ice (10 times). Unbroken cells and large debris were removed by a low speed spin (800g for 10min). The supernatant was centrifuged at 10,000g for 30 min at 4ºC, yielding supernatant (S10) and pellet (P10). Supernatant S10 was collected and centrifuged at 100,000g in 50% sucrose solution for 60min at 4ºC (Beckman 80 Ti rotor ) thereby yielding supernatant S100 and ribosomal pellet P100. Ribosomal pellets contained in P100 fractions was resuspended in a guanidine thiocyanate buffer containing 10% mercaptoethanol (RLT buffer, RNeasy Mini Kit, Qiagen ). RNA was extracted using the RNeasy Mini Kit for isolation of total RNA (Qiagen, Valencia, CA) following the manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE). The purity and integrity of the RNA was monitored by electrophoresis using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Label
CY5
Label protocol
1.0µg of RNA extracted from ribosomal pellets was amplified using the Low RNA input Fluorescent Linear Amplification kit (Agilent Technologies, Palo Alto, A). Briefly RNA was mixed with 1.2 µL of T7 promoter primer and nuclease free water. The reaction mix was incubated at 65°C for 10 minutes and then reactions was placed on ice for 5 minutes. 8.5 µL of cDNA master mix was added to each reaction and incubated at 40°C for two hours. Then the reaction was incubated at 65°C for 15 minutes. 57.6 µL of transcription master mix including the Cyanine 5-CTP (10mM) was added and then incubated at 40°C for 2 hours, to produce the cRNA. The cRNA was purified using the RNeasy mini spin column from Qiagen and dye incorporation was monitored on an Agilent Bioanalyzer.
Hybridization protocol
Hybridization of Cy5 and Cy3 labeled cRNAs were performed using Yeast Oligo Microarrays slides and hybridization kit from Agilent technologies (Sheldon manufacturing, Cornelius, Oregon).To each fluorescent cRNA sample, 2.2 µL of 25X fragmentation buffer was added, then incubated at 60°C for 30 minutes. After the incubation, 55 µL of 2X GEx hybridization buffer were added to the sample. Samples were placed into the array slides. The array slides were placed in the hybridization chambers and incubated at 60°C for 17 hours. Array slides were washed in gene expression wash buffer 1 and 2 and finally acetonitrile as stabilization and drying solution.
Scan protocol
Arrays were scanned with a VersArray Chip Reader system (Bio-Rad, Hercules, CA) at a resolution of 5μm with detector sensitivity values set between 704-800 and laser power at 85%. Scanned images were transferred to the Imagene 3.0 software program (Biodiscovery, El Segundo, CA) for further analysis to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 TIFF files (raw data).
Description
none
Data processing
The microarrays raw data generated with Imagene 3 were analyzed using Limma software (Bioconductor Package 1.7). The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2(M), where M is the log2-fold change after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed [36]. Genes with a p-value ≤ 0.018 was established as a cutoff for differential expression. In addition, a false discovery rate (FDR) [37] was performed with Limma software.
