strain: SREBΔ time point: 24-hrs 22oC replicate: B
Growth protocol
3 biological replicates of SREBΔ and the isogenic wild-type control (ATCC 26199) were grown 37oC and for 6, 24, and 48-hrs at 22oC. All cultures were grown in liquid HMM (Histoplasma macrophage media) containing 10 uM FeSO4. All glassware was pre-treated with 2N HCl and extensively washed with ddH2O.
Extracted molecule
total RNA
Extraction protocol
For all samples, total RNA was extracted using the phenol-guanidinium thiocyanate-1-bromo-3-chloropropane method [Sambrook J and Russell DW. Molecular cloning: a laboratory manual, 3rd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. pp. 7.4 – 7.8.]. B. dermatitidis cells were washed with PBS (pre-warmed to 37oC or 22oC), frozen in liquid nitrogen, lysed by grinding in a mortar and pestle, and treated with TRI Reagent followed by 1-bromo-3-chloropropane (Molecular Research Center Inc., Cincinnati, OH). Total RNA was precipitated using a 1:1 concentration of isopropanol and high salt solution (Molecular Research Center, Inc., Cincinnati, OH), and washed with 75% ethanol. Following resuspension in water pre-treated with diethyl pyrocarbonate (DEPC; Calbiochem, San Diego, CA), total RNA was treated with turboDNAase (Applied Biosystems/Ambion, Austin Texas), and purified using an RNeasy kit (Qiagen, Valencia, CA). To optimally remove guanidine salts, an extra wash of RPE buffer was performed during RNAeasy purification. RNA quality was assessed using an Agilent bioanalyzer (Agilent Technoligies, Santa Clara, CA). Double-stranded cDNA was synthesized and purified at the Gene Expression Center at the University of Wisconsin – Madison. Roche NimbleGen performed Cy3 labeling, hybridization, scanning, and RMA (Robust Multi-Array analysis) normalization for all 24 microarrays at their facility in Reykjavík, Iceland.
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol.
Description
B. dermatitidis SREBΔ incubated for 24-hrs at 22oC. It is the 2nd biological replicate for this time point, each from separate cultures.
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction (NimbleScan software package, version 2.4.27 - Roche NimbleGen, Inc.).
