We prepared RNA from yeast-form Histoplasma capsulatum strain G217B (ATCC 26032; a kind gift of William Goldman, Washington University, St. Louis, MO) under a variety of conditions (including early, middle, and late logarithmic growth, stationary phase, heat shock (42C for 30 min), oxidative stress (1 mM menadione), sulfhydryl reducing stress (10mM DTT), and a range of media (HMM (J. Med. Vet. Mycol. 26:137), 3M (J. Med. Vet. Mycol. 26:137), YPD (Meth. Enz. 194:3), and SD complete (Meth. Enz. 194:3). PolyA RNA were prepared as previously described (Mol. Biol. Cell 14:2314, Mol. Biol. Cell 16:4792).
Label
Cy5
Label protocol
Cy5-labeled cDNA was prepared from individual RNA samples as previously described (Mol. Biol. Cell 16:4792), and an equal mass of cDNA was pooled from each sample and hybridized to individual tiling arrays.
We prepared RNA from mycelial-form Histoplasma capsulatum strain G217B (ATCC 26032; a kind gift of William Goldman, Washington University, St. Louis, MO) in Saboraud or HMM (J. Med. Vet. Mycol. 26:137) shaking liquid cultures or HMM still liquid culture. Total RNA were prepared as previously described (Mol. Biol. Cell 14:2314, Mol. Biol. Cell 16:4792).
Label
Cy3
Label protocol
Cy3-labeled cDNA was prepared from individual RNA samples as previously described (Mol. Biol. Cell 16:4792), and an equal mass of cDNA was pooled from each sample and hybridized to individual tiling arrays.
Hybridization protocol
Fluorescently labeled cDNA was hybridized to CombiMatrix arrays as previously described (Mol. Biol. Cell 16:4792).
Scan protocol
Images of the hybridized arrays were acquired with a GenePix 4000B scanner (Axon Instruments) controlled by the GenePix 4.0 program (Molecular Devices). Each array was scanned using the following PMT settings of 400 and 350 for the 635 and 532 nm lasers respectively.
Data processing
Images were gridded with GenePix 4.0 and the median foreground intensity from the Cy5 channel for each feature was used as the input for subsequent analysis and is reported in the VALUE column (the Cy3 channel was not analyzed due to low signal). Background intensity was estimated based on the median intensities of a control set of known antisense and intergenic regions, a method similar to the use of median intensities of known introns in the analysis of rice tiling data (Nat. Genet. 38:124). Specifically, the background intensity was estimated as the median intensity of the positive control probes corresponding to the intergenic (untranscribed) regions flanking CBP1 and TYR1 and the antisense (untranscribed) probes for CBP1, TYR1, and TEF1. A tiling probe was considered detected if it had intensity greater than the background intensity estimated for the corresponding array.
