title: Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of BALB/c mouse lung infected with B. bronchiseptica strain RB50, and B. bronchiseptica strain RB50 btrS knockout mutant
design: RNA-Seq of BALB/c mouse lung infected with B. bronchiseptica strain RB50, and B. bronchiseptica strain RB50 btrS knockout mutant
library_construction_protocol: Mice were uninfected or intranasally challenged with 30 μl of PBS containing 5x10^5 units of Bordetella bronchiseptica wild type RB50 or mutant RB50ΔbtrS. Mutant RB50ΔbtrS is not able to suppress host immune responses. At day 7 post infection mice were euthanized and lungs were collected in cold PBS. Lungs were collected in 4 different beads tubes containing Trizol (Invitrogen) and kept on ice for the shortest time possible before being homogenized and frozen at -200C.  B. bronchiseptica RB50 and mutant were cultured in Difco Bordet- Gengou (BG) agar (BD, cat. 248200) and supplemented with 10% sheep defibrinated blood (Hemostat) with 20 g/mL streptomycin (VWR Life sciences) or classical LB broth (Difco) as previously described in Gestal et al, Front Microbiol. 2018;9:1969; doi:10.3389/fmicb.2018.01969 Two mouse models were used: BALB/c and the eosinophil deficient pair, ΔdblGATA-1. Mice were originally purchased from Jackson laboratories, Bar Harbor, ME, and then breed under the care of the employees and veterinarians of Louisiana State University Health - Shreveport Animal Care Facility, Shreveport, LA, (AUP:20-038, AUP:22-031). All experiments were carried out in accordance with all institutional guidelines (AUP:20-0038, AUP:22-031). For our animal inoculations mice were anesthetized with 5% isoflurane and when sleep they were intranasally challenged with 30μl of PBS containing either 1x10^5 CFU/mL B. bronchiseptica RB50 or RB50ΔbtrS. For RNA extraction, we followed the protocols recommended by the manufacturer, PureLink RNA Extraction kit, (Invitrogen) and the 4 different tubes were pooled together to have the whole lung RNA represented in one only preparation. We used 3-4 naïve animals and between 5-6 infected mice for each condition. Samples were quantitated with a Qubit RNA assay (ThermoFisher Scientific) and RNA quality was determined with the Agilent TapeStation RNA assay (Agilent Technologies).  One ug of RNA was processed for each sample: mRNA was purified and fragmented. cDNA was synthesized, and 3' ends were adenylated. Anchor sequences were ligated to each sample and limited-cycle PCR was performed to amplify and index the libraries. The average library size (330-355) was determined using an Agilent TapeStation D1000 assay (Agilent Technologies) and libraries were quantitated with qPCR (Bio-rad CFX96 Touch Real-Time PCR, NEB Library Quant Kit for Illumina).