Gene expression profiling in Nlrp3 deficient mice infected with Francisella tularensis

This study investigated gene expression profile in wild type and nlrp3 deficient mice infected with F. tularensis LVS. RNA-sequencing was performed to determine the transcript levels of genes using total RNA extracted from lungs of wild type C57BL/6 and Nlrp3-/- mice infected with 1 × 104 CFU of F. tularensis LVS on days 3 and 7 post-infection. Overall design: Wild type C57BL/6 and Nlrp3-/- mice infected intranasally with 1 × 104 F. tularensis LVS were sacrificed on days 3 and 7 post-infection. Lungs were harvested aseptically from these mice and placed in excess of trizol. Organs were snap-frozen in liquid nitrogen and stored at -80°C until required. When ready, the organs frozen in trizol were thawed on ice and homogenized in 2mL of fresh trizol using a Tissue-tearor. The homogenate was centrifuged at 15,000×g for 15 minutes at 4°C to pellet the tissue debris and limit RNA degradation from tissue-resident nucleases. Total RNA was extracted from the clarified supernatants using the “Purelink RNA mini kit” (Invitrogen) following the manufacturer's instructions. DNA was eliminated from the samples by DNAse treatment using “RNAse-Free DNAse” (Qiagen). Purified RNA was used for RNA-sequencing.