Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
Labeled samples were hybridized to Affymetrix MoGene 1.0 ST GeneChips and scanned with an Affymetrix GeneChip Scanner 3000, generating cell intensity files for each array.
nucleic acid labeling protocol
Labeled cDNA was synthesized from 200 ng total RNAs using NuGEN Applause WT-Amp Plus ST systems (NuGEN Technologies)
nucleic acid extraction protocol
RNAs from the ileal tissue were extracted and purified using classical TRIzol/chloroform protocol. All samples were treated with Turbo DNase (Ambion). RNA quality was determined using Experion Automated Electrophoresis Station (Bio-Rad).
All experiments involving mice were handled in accordance with the Pasteur Institute guidelines for animal welfare. Only 9-12 week-old female C57BL6/J conventional (Charles River) and germfree wild type mice (CDTA) were used for experiments. Germ-free mice were housed in plastic gnotobiotic isolators. L. monocytogenes overnight cultures were diluted in BHI and bacteria were grown until OD600nm=1. Bacterial cultures were centrifuged at 3,500 × g for 15 min. After three washes in PBS, L. monocytogenes pellet was resuspended in PBS at a final concentration of 2.5 × 1010 bacteria/mL. Mice were infected orally with 5 × 109 bacteria diluted in 200 uL of PBS supplemented with 300 uL of CaCO3 (50 mg/mL) for 24 h and 72h. Serial dilutions of the inoculum were plated to control the number of L. monocytogenes inoculated in mice.