Henle cells (Intestine 407 cells; American Type Culture Collection, Manassas, VA) were infected with S. flexneri at an MOI of 100:1 as described previously (Hong M, et al. Infect. Immun. 66:4700–4710). During infection, Henle cells were maintained in Henle medium [Minimum Essential Medium (Invitrogen, Carlsbad, CA) supplemented with 1X nonessential amino acid solution (Invitrogen), and 10% fetal bovine serum (Invitrogen)] in a 5% CO2 atmosphere at 37°C. Gentamycin was added to the media to kill extracellular bacteria at 45 min post-invasion. RNA was isolated 4 hours post-infection
Extracted molecule
total RNA
Extraction protocol
S. flexneri RNA was isolated from infected Henle cells using a modification of the methods described by Eriksson S, et al. 2003 Mol. Microbiol. 47:103–118. Briefly, infected Henle cells were lysed on ice for 30 minutes in 0.1% SDS, 1% acidic phenol, 19% ethanol in water to stabilize bacterial RNA. Cellular lysates were centrifuged, and bacterial pellets were frozen at -80 oC. RNA was subsequently isolated using RNeasy columns (Qiagen). RNA was eluted in 35 µl RNAse free water then treated with DNAse (Ambion) to ensure adequate removal of genomic DNA contamination. RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer and a Nanodrop ND-1000.
Label
Cy5
Label protocol
cDNA was synthesized from RNA using the Invitrogen SuperScript Double-Stranded cDNA synthesis kit. cDNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer and a Nanodrop ND-1000. cDNA labeling with Cy-5 was performed using the NimbleGen labeling kit at the Institute for Bioinformatics and Evolutionary Studies, Genomic Resources Laboratory, University of Idaho, Moscow, ID, USA, (http://cores.ibest.uidaho.edu/genomics-resources-core/services/microarrays) following their standard operating procedures.
Hybridization protocol
Array hybridization was performed at the Institute for Bioinformatics and Evolutionary Studies, Genomic Resources Laboratory, University of Idaho, Moscow, ID, USA, (http://cores.ibest.uidaho.edu/genomics-resources-core/services/microarrays) following their standard operating procedures. Hybridization and washes used NimbleGen reagents
Scan protocol
Scanning with an Roche Nimblegen MS200 scanner in accordance with the protocols provided by NimbleGen was performed at the Institute for Bioinformatics and Evolutionary Studies, Genomic Resources Laboratory, University of Idaho, Moscow, ID, USA, (http://cores.ibest.uidaho.edu/genomics-resources-core/services/microarrays).
Data processing
NimbleScan software (NimbleGen. Madison, WI) was used to align a chip-specific grid to control features and extract raw intensity data for each probe and each array. Chip images were then visually checked for each array and verified not to contain any significant spatial artifacts. Raw intensity data was then read into the R statistical computing environment and checked for quality. Further, chip intensity distributions, boxplots and hierarchical clusters were compared and checked for any unusual global patterns. Each array was then background corrected and normalized using the quantile normalization procedure and finally each probeset was summarized using the median polish procedure as described with the robust multichip average (RMA) procedure (Rafael. A. et al., Nucleic Acids Research 31(4):e15; Irizarry et al. Biostatistics 4(2):249), Bolstad et al. Bioinformatics 19(2):185.
