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
Overall design
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 (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 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.
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.
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