RNAseq of C57BL/6 uninfected lung
Frozen lungs were first macerated with the help of a pestle and pot. To proceed with total RNA extration, TRIzol LS (Thermo Scientific) was used according to the manufacturer's protocol After RNA extraction, the DNA was digested with Rnase-free Dnase (RQ1 Rnase-Free Dnase, Promega Corporation) in the presence of the ribonuclease inhibitor Rnasin (Promega Corporation). The purification was done with the addition of 1 volume of phenol:chloroform (1:1), followed by 100% ethanol:3M sodium acetate (10:1) RNA precipitation. Then, rRNA was depleted using the RiboMinus Eukaryote Kit for RNA-Seq (Thermo Scientific). cDNA library was synthesized using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs) and the NEBNext Multiplex oligos E7335 and E7500 (New England BioLabs). The cDNA library was sequenced in the NGS equipment Brazilian Biorenewables National Laboratory (LNBR), part of the Brazilian Centre for Research in Energy and Materials (CNPEM), a private non-profit organization under the supervision of the Brazilian Ministry for Science, Technology, and Innovations (MCTI)