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:Transscript profiles of Postia placenta grown on different substrates were analyszed. Array design was based on the DoE's Joint Genome Institute's gene models for P. placenta version 1. The research goal is to identtify genes essential for cellulose depolymerization. Keywords: Culture condition comparison From a data set of 12,438 unique alleles, each NimbleGen (Madison, WI) array featured 10 unique 60mers per gene, all in triplicate. The dataset was manually annotated to include only the âbest allelic modelâ among CAZY-encoding genes (SI Table 1, appended as a supplementary file). Total RNA was purified from 6-day old cultures containing microcrystalline cellulose (Avicel) or glucose as sole carbon source. 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 Postia placenta MAD-698 whole genome 37K expression 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 200 units of SuperScript II (Invitrogen) for 60 min at 42Â°C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07 units per ul DNA ligase (Invitrogen), 0.27 units per ul DNA polymerase I (Invitrogen), 0.013 units per ul RNase H (Invitrogen), at 16Â°C for 2 hours. Immediately following, 10 units T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16Â°C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37Â°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 37Â°C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42Â°C. Supplementary file 'si_figure_3.jpg' represents a Neighbor-Joining tree of the cytochrome P450 contingent (P450ome) of P. placenta. The tree shows 254 P450 proteins clustered into 11 fungal clans (indicated by different clan specific color at the branch point). The unrooted phylogenetic tree was constructed using MEGA4 phylogenetic analyses program with 1000 bootstrap replications. Evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset.

Project description:Transscript profiles of Ceriporiopsis subvermispora grown on different substrates were analyszed. Array design was based on the DoE's Joint Genome Institute's gene models for C. subvermispora version 1. The research goal is to identtify genes essential for lignocellulose depolymerization. From a data set of 12,125 unique gene models, each NimbleGen (Madison, WI) array targeted 12083 genes and featured 8 unique 60mers per gene, all in quadruplicate. Total RNA was purified from 5-day old cultures containing microcrystalline cellulose (Avicel) or glucose or ball-milled Populus as sole carbon source. Three biological replicates per medium were used (9 separate arrays). 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 five 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 two volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20 0C. 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 -20C. The RNAs were spun one hour at 2940 x g, washed once with 70% ethanol, and resuspended in 50-100 ?l RNase-free H2O. Three biological replicates per medium were used (nine separate arrays). RNA was converted to double-strand cDNA using Invitrogen's ds cDNA Synthesis kit (Carlsbad, CA) and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10 ?g of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5 mM dNTPs, 100pM 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, 0.2 mM dNTPs, 0.07 U/ul DNA ligase (Invitrogen, Carlsbad, CA), 0.27 U/ul DNA polymerase I (Invitrogen, Carlsbad, CA), 0.013 U/ul RNase H (Invitrogen), at 16°C for two hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for an additional five minute incubation at 16°C. Double-stranded cDNA was treated with 27 ng/ul of RNase A (EpiCentre Technologies, Madison, WI) for 10 min at 37°C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion/Life Technologies, Grand Island, NY), ethanol precipitated, washed with 80% ethanol, and resuspended in 20 ?l water. One ?g of each cDNA sample was amplified and labeled with one unit per ?l of Klenow Fragment (New England BioLabs, UK) and one OD unit of Cy3 fluorophore (TriLink Biotechnologies, Inc., San Diego, CA) for two hours at 37°C. Array hybridization was carried out with 6 ?g of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42°C. Arrays were scanned on the Axon 4000B Scanner (Molecular Devices, Sunnyvale, CA) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v4 (Madison, WI) software was used to quantify and visualize data. .

Project description:Transcript profiles of Postia placenta grown on media containing ball-milled aspen or ball-milled pine as the sole carbon source were analyzed. Array design was based on the DoE's Joint Genome Institute's gene models for P. placenta version 1. The research goal is to identtify genes essential for cellulose depolymerization. From a data set of 12,438 unique alleles, each NimbleGen (Madison, WI) array featured 10 unique 60mers per gene, all in triplicate. The dataset was manually annotated to include only the ‘best allelic model’ among CAZY-encoding genes. Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. 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 Postia placenta MAD-698 whole genome expression 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 200 units 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, 0.07 units per ul DNA ligase (Invitrogen), 0.27 units per ul DNA polymerase I (Invitrogen), 0.013 units per ul RNase H (Invitrogen), at 16°C for 2 hours. Immediately following, 10 units 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.

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Ceriporiopsis subvermispora gene expression in different media

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