Project description:Transcript profiles of Phanerochaete chrysosporium grown on different substrates were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in cellulose degradation. Keywords: gene expression under different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from below mentioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.

Project description:Transcript profiles of Phanerochaete chrysosporium grown on ball-milled aspen or ball-milled pine were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignocellulose degradation. From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and M-BM-< volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20M-KM-^ZC. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturerM-bM-^@M-^Ys protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20M-KM-^ZC. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (6 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42M-KM-^ZC. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16M-KM-^ZC for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16M-KM-^ZC. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37M-KM-^ZC. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37M-KM-^ZC. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42M-KM-^ZC. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.

Project description:Transcript profiles of Phanerochaete chrysosporium grown on different substrates were analyszed. Array design was based on the DoE's Joint Genome Institute's gene models for P. chrysosporium version s. The research goal is to identify genes essential for lignocellulose depolymerization. From a data set of 10004 unique gene models, each NimbleGen (Madison, WI) array targeted 9959 genes and featured 12 unique 60mers per gene, all in triplicate. RNA and protein were obtained from P. chrysosporium strain RP78 (Forest Mycology Center, Forest Products Lab) grown in HighleyM-bM-^@M-^Ys basal salt medium containing 0.5% (w/v) wiley milled (1 mm mesh) wood. For each of the three samples (hybrid line P717, transgenic line 82, transgenic line 64) multiple branches were harvested, debarked and dried. Each two-liter Erlenmeyer flask contained 250 ml medium and was inoculated with approximately 10e7 P. chrysosporium spores scraped from the surface of YMPG agar. Cultures were incubated for five days on a rotary shaker (150 RPM) at 37M-BM-0 C. For RNA, mycelium from triplicate cultures was collected by filtration through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 M-BM-0C until use. For mass spectroscopic analysis, culture filtrates were stored at -20 before use. P. chrysosporium Roche NimbleGen array design was similar to GPL8022, but repetitive elements were removed and 25 gene targets were added. The latter corresponded to open reading frames for which peptides had been detected in culture filtrates. Culture condition compariso: Total RNA was purified from frozen mycelial pellets, converted to Cy3 labeled cDNA, hybridized to microarrays, and scanned as described by Vanden Wymelenberg et al 2010 (Appl Enviro Microbiol 76:3599-3610). The 9 arrays used in these experiments were scanned on the Axon4000B Scanner (Molecular Dynamics) and data extracted using NimbleScan v2.4. Quantile normalization and robust multi-array averaging (RMA) were applied to the raw data using DNASTAR ArrayStar v4 (Madison, WI). Expression levels are based on log2 signals and significant differences in expression were determined using the Moderated t-Test with the FDR threshold set at P<0.05. triplicate cultures for each substrate (wood genotype); The three wood substrates are lines 64, 82 and P717; three different media, and each medium was represented by three flasks (reps).

Project description:Transcript profiles of Phanerochaete chrysosporium grown on carbon-limited medium, nitrogen-limited medium and replete medium. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignin degradation. Keywords: gene expression under three different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from the five abovementioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA; ) were applied to the entire dataset.

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Cellulose-induced regulation of Phanaerochaete chrysosporium genes

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