ABSTRACT: 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. .
