Active Mycobacterium tuberculosis regulatory networks from alveolar macrophages of in vivo mouse infection uncovered by Path-seq

Transcriptional profiling of Mycobacterium tuberculosis mRNA enriched from host-pathogen samples from in vivo alveolar macrophages (Mus musculus). Overall design: A mid log-phase stock of M. tuberculosis H37Rv was used to infect mice in an aerosol infection chamber. At 24 h after infection, bronchoalveolar lavage was performed on euthanized mice. Cells from BAL were filtered, spun down and resuspended in 96-well plate for antibody staining. Cell sorting was performed on a FACS Aria and the entire alveolar macrophage was resuspended in TRIzol (Invitrogen) where total RNA was isolated from mixed host-pathogen sample. MTB from bacterial culture in 7H9 media was also collected at 24 h as a control. Samples were processed by Path-seq, for enrichment of MTB transcripts. The sequence reads that passed quality filters were aligned to a combined M. tuberculosis and M. musuclus genome with Bowtie2 and processed using the R package DuffyNGS.