genotype/variation: rpoE mutant infection: Not applicable condition: Bacteria grown to log phase, pelleted and resuspended in cold LB medium for 16 hours
Treatment protocol
RNA samples were further enriched for bacterial mRNAs using a MICROBExpress kit (Ambion, USA) to deplete bacterial 16S and 23S rRNAs, according to the manufacturer’s instructions. For RNA samples comprising both bacterial and mammalian RNAs, bacterial RNAs were separated from mammalian 18S and 28S rRNAs using a MICROBEnrich kit (Ambion, USA), according to the manufacturer’s instructions. The MICROBEnrich kit uses a Capture Oligo Mix of capture oligonucleotides homologous to mammalian 18S and 28S rRNAs. After processing, bacterial mRNA samples were quantified by spectrophotometry, and electrophoresed in TAE/formamide agarose gels to assess overall mRNA quality and integrity.
Growth protocol
Reference sample used (Cy5) is common to all - Bacteria subjected to growth in LB. Harvested at stationary phase (OD600 = 2.0).
Extracted molecule
total RNA
Extraction protocol
Bacterial cells in liquid cultures were isolated by centrifugation at 3500rpm at 4°C for 15mins, washed with 1xDPBS, and re-pelleted. Cell pellets were mixed with 4-8ml TRIZOL reagent (Invitrogen, USA) and maintained at room temperature for 10mins. For bacterial cells on plate cultures, bacterial colonies were harvested using sterile inoculation loops directly into 4ml TRIZOL. One-tenth the volume of 1-bromo-3-chloro-propane (Sigma, USA) was then added, and the entire mixture vortexed for 15s. The mixture was incubated at room temperature for 3mins and centrifuged at 9,000rpm at 4°C for 30mins. The aqueous phase was transferred to a fresh tube containing an equal volume of isopropanol, mixed well, and maintained at room temperature for 10mins. The tube was then centrifuged at 9,000rpm at 4°C for 30mins to obtain the total RNA pellet. The pellet was washed with 1ml 75% ethanol, dried at room temperature for 5mins, and resuspended in 30ul of RNase-free water. Bp-infected RAW264.7 cells were lysed with TRIZOL and both murine and bacterial total RNAs were extracted using a PureLink RNA mini kit (Invitrogen, USA). Bp-infected murine lungs from infected BALB/c mice were treated with 2ml TRIZOL, homogenized using a Polytron homogenizer (maximum speed, two bursts 10–15s each), and processed for RNA extraction. Extracted RNAs were treated with DNase I (DNA-free; Ambion, USA) to remove contaminating genomic DNA, according to the manufacturer’s instructions.
Label
Cy3
Label protocol
Sample cDNAs were labeled with Cy3 (Cy3-ULS, Kreatech Diagnostics, USA) and a universal hybridization reference (K9LBS) was similarly labeled with Cy5.
Hybridization protocol
Microarray hybridizations were performed at 65°C in a MAUI hybridization station (BioMicro Systems, USA) for 17hrs.
Scan protocol
The slides were washed, dried in a centrifuge at 600rpm for 4mins, and scanned on an Axon Genepix 4000B scanner (Molecular Devices, USA) at a resolution of 5 microns
Description
SAMPLE 1
Data processing
The raw data (.pair file) was subjected to Lowess within-chip normalization as implemented in GeneSpring GX11 , and cross-chip median normalization using R, .
