Frozen cell pellets were suspended in 1 ml of Trizol reagent (GIBCOy BRL) and transferred to 2-ml screw-cap tubes containing 0.4-ml of 0.1-mm diameter zirconiaysilica beads (BioSpec Products, Bartlesville, OK).
Growth protocol
Escherichia coli JM109 was routinely used in DNA-cloning procedures. M. tuberculosis strain H37Rv (American Type Culture Collection) was maintained in Middlebrook 7H9 broth or on 7H10 agar (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-NaCl complex (ADN). When media with defined amounts of iron were needed, 7H9 and 7H10 were prepared omitting ferric ammonium citrate. We refer to these media as reconstituted 7H9 (r7H9) or r7H10. These media were subsequently supplemented with the desired amount of iron in the form of FeCl3. Minimal medium (MM) was also used to grow cultures under defined iron conditions for the determination of mycobactin, growth curves (MM agar), and RNA extraction (MM broth). This medium contained 0.5% (wt/vol) asparagine, 0.5% (wt/vol) KH2PO4, 2% glycerol, 0.5 mg of ZnCl2/liter, 0.1 mg of MnSO4/liter, and 40 mg of MgSO4/liter. It was supplemented with the desired concentrations of FeCl3, and in the case of the broth, 0.05% Tween 80 and 10% ADN were included. Where indicated, antibiotics were included at the following concentrations: kanamycin, 20 ug/ml; streptomycin, 20 ug/ml; and hygromycin, 150 ug/ml.
Extracted molecule
total RNA
Extraction protocol
Cells were disrupted by three 30-sec pulses in a BioSpec Products bead beater. After 5 min at room temperature, samples were centrifuged at 13,000 x g for 45 sec, and the supernatants were extracted with 300 ul of chloroformyisoamyl alcohol (24:1) and precipitated with isopropyl alcohol. Samples were incubated 10 min to overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1 ml of 75% ethanol, centrifuged at 13,000 x g for 5 min, and air-dried. After suspension of the RNA pellets in 90 ul of water, 10 ul of DNase I 10x buffer and 4 units of DNase I (Ambion, Austin, TX) were added and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (Qiagen, Chatsworth, CA).
Label
Cy3
Label protocol
All steps in the MTB DNA microarray gene expression analysis were performed as described. Briefly: 1. Bring 0.5-5 µg RNA (Typically 3 µg) to 11 µl and add 2 µl (~2 mg/ml) random hexamers. 2. Heat 10 min at 65C (or 2 min at 98C), snap cool on ice. 10 rxns 20 rxns. 3. Add 11µl 5.0 µl 5X First-Strand buffer (51) (102) 2.5 µl 100 mM DTT (25.5) (51) 2.0 µl dNTP (5 mM A,G,C and 0.2 mM T) (20.5) (41) 1.5 µl Cy3 or Cy5 (Typically use Cy5 for sample that should change) (15.3) (30.6) Then add 1.2-1.8 µl Stratascript or 0.8-1.2 µl Superscript II RTase. 4. Incubate 10m at 25C followed by 90m at 42C in PCR machine.
Channel 2
Source name
Cell culture grew in iron-sufficient (50 uM FeCl3)
Frozen cell pellets were suspended in 1 ml of Trizol reagent (GIBCOy BRL) and transferred to 2-ml screw-cap tubes containing 0.4-ml of 0.1-mm diameter zirconiaysilica beads (BioSpec Products, Bartlesville, OK).
Growth protocol
Escherichia coli JM109 was routinely used in DNA-cloning procedures. M. tuberculosis strain H37Rv (American Type Culture Collection) was maintained in Middlebrook 7H9 broth or on 7H10 agar (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-NaCl complex (ADN). When media with defined amounts of iron were needed, 7H9 and 7H10 were prepared omitting ferric ammonium citrate. We refer to these media as reconstituted 7H9 (r7H9) or r7H10. These media were subsequently supplemented with the desired amount of iron in the form of FeCl3. Minimal medium (MM) was also used to grow cultures under defined iron conditions for the determination of mycobactin, growth curves (MM agar), and RNA extraction (MM broth). This medium contained 0.5% (wt/vol) asparagine, 0.5% (wt/vol) KH2PO4, 2% glycerol, 0.5 mg of ZnCl2/liter, 0.1 mg of MnSO4/liter, and 40 mg of MgSO4/liter. It was supplemented with the desired concentrations of FeCl3, and in the case of the broth, 0.05% Tween 80 and 10% ADN were included. Where indicated, antibiotics were included at the following concentrations: kanamycin, 20 ug/ml; streptomycin, 20 ug/ml; and hygromycin, 150 ug/ml.
Extracted molecule
total RNA
Extraction protocol
Cells were disrupted by three 30-sec pulses in a BioSpec Products bead beater. After 5 min at room temperature, samples were centrifuged at 13,000 x g for 45 sec, and the supernatants were extracted with 300 ul of chloroformyisoamyl alcohol (24:1) and precipitated with isopropyl alcohol. Samples were incubated 10 min to overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1 ml of 75% ethanol, centrifuged at 13,000 x g for 5 min, and air-dried. After suspension of the RNA pellets in 90 ul of water, 10 ul of DNase I 10x buffer and 4 units of DNase I (Ambion, Austin, TX) were added and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (Qiagen, Chatsworth, CA).
Label
Cy5
Label protocol
same as channel 1
Hybridization protocol
The quality of cDNA labeling was monitored by quantitatively measuring the intensities of Cy3- and Cy5-labeled samples hybridized to the MTB genomic positive control spots.
Scan protocol
Microarrays were scanned using a GenePix 4000 A (Axon Instruments). The intensities of the two dyes at each spot were quantified using SCANALYZE (M. Eisen; http://rana.lbl.gov/EisenSoftware.htm).
Description
Biological replicate 6 of 6. Comparison between ST52 (H37Rv ideR::aph, attB::ideR) and ST22 (H37Rv ideR::aph). Growth conditions iron-sufficient (50 uM FeCl3)
Data processing
The intensities of the two dyes at each spot were quantified using SCANALYZE, written by M. Eisen at Stanford University and available at http://rana.stanford.edu/software. Data processing value is Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm). The overall reproducibility, both biological and technical, of the microarray experiments was evaluated using the Significance Analysis of Microarrays (SAM) program. Six DNA microarray experiments, which compared identical RNAs on the same array, were compared to six microarray experiments on three biological sample sets for each condition. The SAM algorithm was set for two-class unpaired analysis with 500 permutations and K-nearest imputer for missing data. Significantly regulated genes were selected by adjusting the delta value to give a false discovery rate below 1%. In each data set, all genes regulated 1.5-fold and greater were determined to be significant with a false discovery rate of less than 1%, indicating a high degree of reproducibility.
