50 ml culture was rapidly frozen in liquid nitrogen and lyophilized overnight. The lyophilized cells were subsequently used for total RNA isolation. As biological replicates for DNA microarrays, three independent cultures for each strain were prepared for total RNA isolation.
Growth protocol
The wild-type H99 strain and PH3:HSF1 (YSB2200) strain were grown in 50 ml YPD medium at 30°C for 16 h. Then 2.5 ml of the overnight culture was inoculated into 50 ml of fresh YPD medium and further incubated for 4 h at 30°C to an optical density at 600 nm (OD600 =1.0)
Extracted molecule
total RNA
Extraction protocol
For total RNA isolation, the lyophilized cell pellets were added to 3 ml volume of sterile 3 mm glass bead (SIGMUND LINDER), homogenized by shaking, added to 4 ml of TRizol reagent (Tri reagent, Molecular Research Center), and allowed to incubate at room temperature for 5 min. Then 800 µl of chloroform was added, incubated for 3 min at room temperature, transferred to 14 ml round-bottom tubes (SPL), and centrifuged by 10,000 rpm at 4°C for 15 min (Sorvall SS-34 rotor). Two milliliters of the supernatant was transferred to a new round-bottom tube, 2 ml isopropanol was added, inverted several times, and allowed to incubate for 10 min at room temperature. Then the mixture was re-centrifuged at 10,000 rpm at 4°C for 10 min, and the pellet was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC)-treated water and centrifuged at 8,000 rpm at 4°C for 5 min. The pellet was dried and resuspended with 500 µl DEPC-treated water. Concentration and purity of total RNA samples were calculated by measuring OD260nm and gel-electrophoresis, respectively. For control total RNA, all total RNAs prepared from WT and PH3:HSF1 cells grown in conditions described above were pooled (pooled reference RNAs).
Label
Cy3
Label protocol
For cDNA synthesis, the total RNA concentration was adjusted to 1 µg/µl with DEPC-treated water, and 15 µl of the total RNA (15 µg) was added to 1 µl of 5 µg/µl oligo dT (5’-TTTTTTTTTTTTTTTTTTTTV-3’)/pdN6 (Amersham, 1:1 mixture of 10 µg/µl, respectively), incubated at 70°C for 10 min, and place on ice for 10 min. Then 15 µl of cDNA synthesis mixture (3 µl 0.1 M DTT, 0.3 µl RNase inhibitor (Intron), 1.2 µl aa-dUTP (5-(3-aminoallyl)-2’-deoxyuridine 5’-triphosphate)/dNTPs (a mixture of 6 µl dTTP (100 mM), 4 µl aa-dUTP (100 mM), 10 µl dATP (100 mM), 10 µl dCTP (100 mM), 10 µl dGTP (100 mM). 40 µl DEPC-treated water), 1.5 µl RevertAidTM H Minus M-MuLV Reverse Transcriptase (Fermentas), 6 µl RevertAidTM H Minus M-MuLV Reverse Transcriptase buffer, 3 µl DEPC-treated water) was added and incubated at 42°C for 2 hrs. Then 10 µl of 1 N NaOH and 10 µl of 0.5 M EDTA (pH 8.0) were added and incubated at 65°C for 15 min. After incubation, 25 µl of 1 M HEPES buffer (pH 8.0) and 450 µl of DEPC-treated water were added, and the whole mixture was concentrated through a Microcon30 filter (Millipore) and vacuum-dried for 1 hr. For Cy3 and Cy5 (Amersham) labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 µl DMSO and 1 µl of each dye was aliquoted into separate tubes. The cDNAs prepared as described above were added to 9 µl of 0.05 M Na-bicarbonate (pH 8.0) and incubate at room temperature for 15 min. The cDNAs prepared from pooled reference RNAs were mixed with Cy5 as a control and the cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy3. Each mixture was further incubated at room temperature for 1 hr in the dark and purified by QIAquick PCR purification kit (QIAGEN).
50 ml culture was rapidly frozen in liquid nitrogen and lyophilized overnight. The lyophilized cells were subsequently used for total RNA isolation. As biological replicates for DNA microarrays, three independent cultures for each strain were prepared for total RNA isolation.
Growth protocol
The wild-type H99 strain and PH3:HSF1 (YSB2200) strain were grown in 50 ml YPD medium at 30°C for 16 h. Then 2.5 ml of the overnight culture was inoculated into 50 ml of fresh YPD medium and further incubated for 4 h at 30°C to an optical density at 600 nm (OD600 =1.0)
Extracted molecule
total RNA
Extraction protocol
For total RNA isolation, the lyophilized cell pellets were added to 3 ml volume of sterile 3 mm glass bead (SIGMUND LINDER), homogenized by shaking, added to 4 ml of TRizol reagent (Tri reagent, Molecular Research Center), and allowed to incubate at room temperature for 5 min. Then 800 µl of chloroform was added, incubated for 3 min at room temperature, transferred to 14 ml round-bottom tubes (SPL), and centrifuged by 10,000 rpm at 4°C for 15 min (Sorvall SS-34 rotor). Two milliliters of the supernatant was transferred to a new round-bottom tube, 2 ml isopropanol was added, inverted several times, and allowed to incubate for 10 min at room temperature. Then the mixture was re-centrifuged at 10,000 rpm at 4°C for 10 min, and the pellet was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC)-treated water and centrifuged at 8,000 rpm at 4°C for 5 min. The pellet was dried and resuspended with 500 µl DEPC-treated water. Concentration and purity of total RNA samples were calculated by measuring OD260nm and gel-electrophoresis, respectively. For control total RNA, all total RNAs prepared from WT and PH3:HSF1 cells grown in conditions described above were pooled (pooled reference RNAs).
Label
Cy5
Label protocol
For cDNA synthesis, the total RNA concentration was adjusted to 1 µg/µl with DEPC-treated water, and 15 µl of the total RNA (15 µg) was added to 1 µl of 5 µg/µl oligo dT (5’-TTTTTTTTTTTTTTTTTTTTV-3’)/pdN6 (Amersham, 1:1 mixture of 10 µg/µl, respectively), incubated at 70°C for 10 min, and place on ice for 10 min. Then 15 µl of cDNA synthesis mixture (3 µl 0.1 M DTT, 0.3 µl RNase inhibitor (Intron), 1.2 µl aa-dUTP (5-(3-aminoallyl)-2’-deoxyuridine 5’-triphosphate)/dNTPs (a mixture of 6 µl dTTP (100 mM), 4 µl aa-dUTP (100 mM), 10 µl dATP (100 mM), 10 µl dCTP (100 mM), 10 µl dGTP (100 mM). 40 µl DEPC-treated water), 1.5 µl RevertAidTM H Minus M-MuLV Reverse Transcriptase (Fermentas), 6 µl RevertAidTM H Minus M-MuLV Reverse Transcriptase buffer, 3 µl DEPC-treated water) was added and incubated at 42°C for 2 hrs. Then 10 µl of 1 N NaOH and 10 µl of 0.5 M EDTA (pH 8.0) were added and incubated at 65°C for 15 min. After incubation, 25 µl of 1 M HEPES buffer (pH 8.0) and 450 µl of DEPC-treated water were added, and the whole mixture was concentrated through a Microcon30 filter (Millipore) and vacuum-dried for 1 hr. For Cy3 and Cy5 (Amersham) labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 µl DMSO and 1 µl of each dye was aliquoted into separate tubes. The cDNAs prepared as described above were added to 9 µl of 0.05 M Na-bicarbonate (pH 8.0) and incubate at room temperature for 15 min. The cDNAs prepared from pooled reference RNAs were mixed with Cy5 as a control and the cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy3. Each mixture was further incubated at room temperature for 1 hr in the dark and purified by QIAquick PCR purification kit (QIAGEN).
Hybridization protocol
Microarray Hybridization and Washing. A C. neoformans serotype D 70-mer microarray slide containing 7,936 probes (Duke University) was pre-hybridized at 42°C in 60 ml of pre-hybridization buffer (42.4 ml sterile distilled water, 2 ml 30% BSA (Sigma), 600 µl 10% SDS, 15 ml 20x SSC (Saline-Sodium Citrate, 3 M NaCl, 0.3 M Na-citrate, pH 7.0)), washed with distilled water and isopropanol, and dried by brief centrifugation (1000rpm, 2 min). The Cy3- and Cy5-labeled cDNA samples were combined, concentrated through a Microcon30 filter, and vacuum-dried. The dried cDNA samples were resuspended with 24 µl of 1x hybridization buffer (250 µl 50% formamide, 125 µl 20x SSC, 5 µl 10% SDS, 120 µl dH2O, total 500 µl), added with 1 µl polyA tail DNA (Sigma), further incubated at 100°C for 3 min and allowed to cool for 5 min at room temperature. The microarray slides were aligned into the hybridization chamber (DieTech), removed of any dust, and covered by Lifterslips (Erie Scientific). The Cy3/Cy5-labeled cDNA samples were applied in between Lifterslips and slides. To prevent slides from drying, 5 ml of 3x SSC buffer was applied into the hybridization chamber, which were subsequently incubated for 16 hr at 42°C. After incubation, the microarray slides were washed with three different washing buffers (wash buffer 1 (10 ml 20x SSC, 600 µl 10% SDS, 189.4 ml dH2O, preheated at 42°C), wash buffer 2 (3.5 ml 20x SSC, 346.5 ml dH2O), wash buffer 3 (0.88 ml 20x SSC, 349.12 ml dH2O)) for 2, 5, and 5 min, respectively, on an orbital shaker. Three independent DNA microarrays with 3 independent biological replicates were performed.
Scan protocol
After hybridization and washing, the microarray slides were scanned with a GenePix 4100A scanner (Axon Instrument) and the signals were analyzed with GenePix Pro (Ver. 4.0) and gal file (http://genome.wustl.edu/activity/ma/cneoformans).
Description
WT_rep3
Data processing
Since total RNAs isolated from serotype A C. neoformans strains were hybridized on the microarray slides printed with the serotype D 70-mer oligonucleotide sequences, the serotype A gene sequences were mapped to those of the serotype D using BLASTN. C. neoformans H99 gene sequences that were updated at 24 November 2008 were downloaded from Broad institute (http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans). The serotype D C. neoformans JEC21 gene sequences that were updated 27 April 2005 were downloaded from JCVI (ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/c_neoformans/). The functional category of each C. neoformans H99 gene was assigned using the NCBI KOG database (http://www.ncbi.nlm.nih.gov/COG/grace/shokog.cgi). Using the serotype A gene sequence, each S. cerevisiae gene name or ID listed in the supplementary Tables was identified by blastp search (e-value cut-off: e-6). For hierarchical and statistical analysis, data transported from GenePix software were analyzed with Acuity (Axon) by employing Lowess normalization (Lowess M log ratio (F532 Median - B532, F635 Median - B635)), reliable gene filtering, clustering (standard correlation and average linkage) and zero-transformation, and ANOVA analysis (P<0.05), and Microsoft Excel software (Microsoft).
