Ion Torrent Proton sequencing; RNA-Seq of mouse lung vaccinated with subcutaneous BCG and two novel tuberculosis mucosal boosting nanovaccines
BAL was collected by irrigating lungs via trachea with a syringe containing 1mL of cold PBS. Lungs and spleen were aseptically removed after BAL lavage and were homogenized and processed for immunological assays. Prior to homogenization, lungs were incubated in digestion medium DMEM (Dulbecco's Modified Eagle Medium, High glucose NEAA, no glutamine, Gibco) supplemented with collagenase 0.15 mg/mL (Sigma Aldrich) at 37 °C for 30 min. Spleens and collagenase-incubated lungs were homogenized and filtered through a 40 μm nylon mesh cell strainer (BD Biosciences, San Diego, CA) to obtain a homogenous cell suspension. BAL, lung and spleen cells were treated with red blood cell (RBC) lysing buffer (0.87% of NH4Cl solution and 5% of PBS in water) for 5 min, and washed twice with DMEM supplemented with 10% of heat-inactivated fetal bovine serum. Leukocyte fraction was isolated by density gradient centrifugation on an 80%/40% Percoll (GE Healthcare, Sigma Aldrich) gradient.  Mice were vaccinated with 0.5 million CFU of BCG strain Pasteur. Twelve and 14 weeks later, they were anesthetized with 100 uL of ketamine–xylazine and 50 uL of Nano-FP1 nanovaccine was intranasally administered (combination of 0.1 % yellow carnauba palm wax with sodium myristate (YC-NaMA) nanoparticles (NPs) (Bethlehem, PA, USA); 200 µg/mL of a fusion protein (FP1) composed of an N-terminal histidine tag and the Mtb antigens Acr (Rv2031c), Ag85B (Rv1886c), and the heparin-binding domain of HBHA (Rv0475); and 400 ug/ml PolyI:C (Sigma Aldrich) in saline solution. Mice were sacrificed 28 weeks after BCG priming, 14 weeks after second nanovaccine boosting. RNA from BAL cells and immune lung infiltrate was extracted using RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's recommendations. For early response experiments, RNA was extracted from the post-caval lung lobes using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer's recommendations. RNA quality was assessed based on RIN value using Agilent 2100 Bioanalyzer and the Agilent RNA 600 Nano Kit (Agilent Technologies). Three samples of pooled RNA were analysed per condition, selecting those with higher RIN value and RNA concentration. In the case of mice sacrificed at 11 and 14 weeks, two BAL samples were analysed due to low RNA amount. RNA sequencing was performed on an Ion Proton sequencer (Ion Torrent, Thermo Fisher Scientific; CA, USA) at the Genomic Service at the Centro de Apoio Científico-Tecnolóxico á Investigación of University of Vigo (CACTI). Poly(A)-mRNA fraction was enriched using the Dynabeads® mRNA DIRECTTM Micro Kit (Thermo Fisher Scientific). Enriched mRNA was processed with the Ion Total RNA-Seq Kit v2 (Life technologies-Thermo Fisher Scientific). cDNA libraries with a percentage of DNA in 50-160 bp less than 50% passed the quality control and were then used for template preparation.