Conidia were extracted in water from N. crassa strains streaked onto 3 mL agar slants made with Vogel's Medium with 2% sucrose and grown for 7-10 days at room temperature with constant exposure to indoor light. An estimate of conidia concentration was made by taking a sample, diluting 1:40 into water, and measuring the optical density at 530 nm. An OD530 of 0.25 is approximately 108 conidia/mL in the undiluted sample. Conidia were added to a final concentration of 106 conidia per mL into 25 mL of Vogel's Medium with 2% glucose as the carbon source. Cultures were shaken at 200 rpm in a 30°C incubator with lights on. After 8 hours ~100% exhibited hyphal growth with the bulk of germ tube lengths between 50 and 400 µm. At this point mycelia were collected by vacuum filtration. Material was scraped from the filter and placed into tubes containing 0.5 mL of buffer AE (50 mM sodium acetate, 10 mM EDTA), 33.3 µL of 25% SDS, and 0.5 mL of acid phenol:chloroform pH 4.5 (Ambion AM9720) then inverted to mix and flash frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated by the method of hot acid phenol/chloroform extraction. Samples were placed at 65°C in a thermomixer shaking at 1400 rpm for 10 minutes, vortexed for 10 seconds, then placed back in thermomixer for another 5 minutes. Samples were cooled on ice for 5 minutes, then spun down at 12,000 rpm in a microcentrifuge for 15 minutes. The aqueous phase was extracted and placed into a 2 mL phase-lock gel tube. Two more extractions with acid phenol:chlorofom were performed, followed by an extraction with chloroform. RNA was precipitated by adding 1/10th volume of 3M sodium acetate, mixing, then adding 1 volume of isopropanol. Samples were mixed and placed at -20°C for at least an hour. Samples were spun down for 20 minutes at top speed in a microcentrifuge. The RNA pellet was washed with ice-cold 75% ethanol then air dried for 10 minutes before being resuspended in 100 µL of water. RNA yields were ~250 µg.
Label
Cy5
Label protocol
15 µg of RNA was used in each reverse transcription reaction RNA was reverse transcribed in the presence of 5-(3-Aminoallyl)-dUTP. 13.8 µL of sample RNA plus water was mixed with 1 µL of control RNA (Ambion AM1780) and 2 µL of N9 and dT20VN primers (each at 2.5 µg/µL). This mixture was heated to 70°C for 2 minutes then cooled to 4°C. 6 µL of 5x 1st Strand Buffer, 1.2 µL of 25x dNTP/aminoallyl-dUTP mix (Ambion AM8439), 3 µL of 0.1 M DTT, 1 µL SuperaseIn (Ambion AM2696), and 2 µL of Superscript III (Invitrogen 18080-085) was added as a 13.2 µL master mix, and reverse transcription performed at 42°C for 2 hours. RNA was then hydrolyzed by addition of 15 µL of 1 M NaOH and heating to 70°C for 15 minutes. The sample was neutralized by addition of 15 µL of 1 M HCl and 10 µL of sodium acetate pH 5.2. cDNA was purified using the Qiagen MinElute kit and eluted off the column with 20 µL of 10 mM sodium phosphate pH 8.5. Experimental sample cDNA was labeled with Cy5 dye while cDNA made from the wild-type strain was labeled with Cy3 dye (GE Healthcare Life Science RPN5661). A tube of NHS-monoester Cy dye was resuspended in 60 µL of DMSO then 20 µL was used for each appropriate sample. Coupling was performed at room temperature in the dark for 1-2 hours. The labeling reaction was quenched by addition of 9 µL of 3 M hydroxylamine and incubation for 15 minutes. Labeled cDNA was purified using the Qiagen MinElute kit.
Conidia were extracted in water from N. crassa strains streaked onto 3 mL agar slants made with Vogel's Medium with 2% sucrose and grown for 7-10 days at room temperature with constant exposure to indoor light. An estimate of conidia concentration was made by taking a sample, diluting 1:40 into water, and measuring the optical density at 530 nm. An OD530 of 0.25 is approximately 108 conidia/mL in the undiluted sample. Conidia were added to a final concentration of 106 conidia per mL into 25 mL of Vogel's Medium with 2% glucose as the carbon source. Cultures were shaken at 200 rpm in a 30°C incubator with lights on. After 8 hours ~100% exhibited hyphal growth with the bulk of germ tube lengths between 50 and 400 µm. At this point mycelia were collected by vacuum filtration. Material was scraped from the filter and placed into tubes containing 0.5 mL of buffer AE (50 mM sodium acetate, 10 mM EDTA), 33.3 µL of 25% SDS, and 0.5 mL of acid phenol:chloroform pH 4.5 (Ambion AM9720) then inverted to mix and flash frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated by the method of hot acid phenol/chloroform extraction. Samples were placed at 65°C in a thermomixer shaking at 1400 rpm for 10 minutes, vortexed for 10 seconds, then placed back in thermomixer for another 5 minutes. Samples were cooled on ice for 5 minutes, then spun down at 12,000 rpm in a microcentrifuge for 15 minutes. The aqueous phase was extracted and placed into a 2 mL phase-lock gel tube. Two more extractions with acid phenol:chlorofom were performed, followed by an extraction with chloroform. RNA was precipitated by adding 1/10th volume of 3M sodium acetate, mixing, then adding 1 volume of isopropanol. Samples were mixed and placed at -20°C for at least an hour. Samples were spun down for 20 minutes at top speed in a microcentrifuge. The RNA pellet was washed with ice-cold 75% ethanol then air dried for 10 minutes before being resuspended in 100 µL of water. RNA yields were ~250 µg.
Label
Cy3
Label protocol
15 µg of RNA was used in each reverse transcription reaction RNA was reverse transcribed in the presence of 5-(3-Aminoallyl)-dUTP. 13.8 µL of sample RNA plus water was mixed with 1 µL of control RNA (Ambion AM1780) and 2 µL of N9 and dT20VN primers (each at 2.5 µg/µL). This mixture was heated to 70°C for 2 minutes then cooled to 4°C. 6 µL of 5x 1st Strand Buffer, 1.2 µL of 25x dNTP/aminoallyl-dUTP mix (Ambion AM8439), 3 µL of 0.1 M DTT, 1 µL SuperaseIn (Ambion AM2696), and 2 µL of Superscript III (Invitrogen 18080-085) was added as a 13.2 µL master mix, and reverse transcription performed at 42°C for 2 hours. RNA was then hydrolyzed by addition of 15 µL of 1 M NaOH and heating to 70°C for 15 minutes. The sample was neutralized by addition of 15 µL of 1 M HCl and 10 µL of sodium acetate pH 5.2. cDNA was purified using the Qiagen MinElute kit and eluted off the column with 20 µL of 10 mM sodium phosphate pH 8.5. Experimental sample cDNA was labeled with Cy5 dye while cDNA made from the wild-type strain was labeled with Cy3 dye (GE Healthcare Life Science RPN5661). A tube of NHS-monoester Cy dye was resuspended in 60 µL of DMSO then 20 µL was used for each appropriate sample. Coupling was performed at room temperature in the dark for 1-2 hours. The labeling reaction was quenched by addition of 9 µL of 3 M hydroxylamine and incubation for 15 minutes. Labeled cDNA was purified using the Qiagen MinElute kit.
Hybridization protocol
Labeled cDNA from an experimental and wild-type sample were mixed (27 µL total) along with 6 µL of 20X SSC (1X SSC is 150 mM NaCl, 15 mM sodium citrate at pH 7.0), 2 µL of Qiagen buffer EB (10 mM Tris-HCl, pH 8.5), 3 µL of 10 µg/µL polyA RNA (Sigma P4303), 1 µL of 1 M Hepes-NaOH pH 7.0, and 1 µL of 10% SDS. This 40 µL probe mixture was heated at 95°C for 2 minutes then centrifuged for 5 minutes. Arrays were post-processed on the day of hybridization. Arrays were rehydrated by placing slides face down over 50 mL of 0.5X SSC in a humidity chamber (Sigma H6644) for 30 minutes. Arrays were then snapped dried on a 70-80°C inverted heat block for 5 seconds. For N. crassa arrays, the DNA was crosslinked to the slide using 600 millijoules of UV energy in a Stratalinker. The aminosilane surface was blocked by incubation for 35 minutes in a solution of 5X SSC, 1% SDS, and 1% w/v of Blocking Reagent (Roche 11096176001) at 60°C. Arrays were washed twice in high-quality water for 2 minutes at room temperature then dried by centrifugation Probe mixture containing labeled cDNA was hybridized to post-processed microarrays using the MAUI hybridization system (BioMicro) at 65°C for ~16 hours.
Scan protocol
The MAUI mixer was removed from the microarray while submerged in a warm solution of 2X SSC and 0.01% SDS. The array was then placed in a 2X SSC solution at room temperature until all arrays were ready for washing. Arrays were washed with 2X SSC and 0.05% SDS at 65°C for 5 minutes with agitation, then at room temperature with agitation in 2X SSC for 1 minute, another 2X SSC wash for 2 minutes, 1X SSC for 2 minutes, and 0.2X SSC for 2 minutes. The arrays were dried by centrifugation in a low-ozone environment. Microarrays were scanned using an AxonScanner 4000B and GenePix 6.0 software (Molecular Devices). PMT levels were set to maximize signal in each channel and only saturate a few spots.
Description
NA
Data processing
Spots were located using the GenePix software with some manual adjustment and flagging. Spots were then auto-flagged as bad if they met any of the following criteria: greater than 10% of the spot pixels were saturated in either channel, the spot contained 12 pixels or less, the R2 for the fit between Cy5 (red) and Cy3 (green) pixel intensities was less than 0.6, or if in either channel the signal intensity minus the local background is less than three times the standard deviation of the local background. Array data were exported in a GenePix Results file (.gpr) and further processed and analyzed within the R statistical environment. After data were loaded into R, a spot was filtered if signal intensity was not 2x over background in both channels for N. crassa gene expression experiments. For features passing flagging and filtering, we calculated the ratio between the experiment and reference channels as log2(red signal - red background)/(green signal - green background)). Log2 ratio data for each experiment were mean centered and then replicate spots on the array were averaged.
