Illumina HiSeq 2500 sequencing; RNA-Seq of murine lung tissue and blood treated with S. pneumoniae TIGR4 or S. aureus USA300 against PBS controls
RNA-Seq of murine lung tissue and blood treated with S. pneumoniae TIGR4 or S. aureus USA300 against PBS controls
The S. pneumoniae strain TIGR4 was grown at 37°C in Todd-Hewitt broth supplemented with 1% (w/v) yeast extract (THY-medium) and 1% of heat-inactivated fetal calf serum. Bacteria were grown to the Mid-Log phase (OD600 equal to app. 0.4), collected by centrifugation at 4000 rpm for 10 min and washed once with sterile phosphate-buffered saline (PBS). The methicillin-resistant S. aureus strain USA300 was grown at 37°C in Tryptic Soy broth (TSB) with shaking (250 rpm) overnight. Fresh TSB was inoculated with the overnight culture and grown to the Mid-Log phase (OD600 equal to app. 0.5) at 37°C and 250 rpm. Aliquots of 50 ml culture were centrifuged at 4000 rpm for 15 min at 4°C and washed once with sterile PBS. For intranasal infection, mice were anesthetized by intraperitoneal injection with ketamine-xylazine solution and inoculated intranasally with either 5x10^7 CFU of S. pneumoniae TIGR4 in 20 microL of PBS,5x10^8 CFU of S. aureus USA300 in 20 microL of PBS or with 20% L of PBS as mock treatment. Lung tissue and blood were collected at two time points, 24 h and 48 h post infection. For RNA isolation from lungs, lung tissue was washed in PBS and incubated in 2 mL of RNAlater overnight at 4°C to stabilize RNA. Total RNA was isolated from lung tissue samples using the miRNeasy Mini Kit (Qiagen) following the manufacturer's instructions. For RNA isolation from blood, a volume of 100 microL of blood was collected in RNAprotect Animal Blood Tubes and kept for at least 2 h at room temperature before cold storage. Total RNA was isolated from the blood samples using the RNeasy Protect Animal Blood Kit (Qiagen) according to the manufacturer's instructions. Similar amounts of RNA extracted from lung tissue of 2 to 3 mice per experiment and from blood of 4 to 6 mice per experiment were pooled. In total, 3 RNA pools (biological replicates) for each condition, organ and time point were generated. Libraries from lung tissue and blood RNA-Seq were generated using the ScriptSeq v2 RNA- Seq library preparation kit (Illumina) according to the manufacturer's instructions.
Experimental Factor: organism part
lung
Experimental Factor: infect
Streptococcus pneumoniae TIGR4
Experimental Factor: time
24