An optimized protocol for dual RNA-Seq of human macrophages infected with Mycobacterium avium

Recently, dual RNA-sequencing (RNA-Seq) has been identified as a powerful tool to study host-pathogen interactions, which is particularly interesting for intracellular pathogens such as mycobacteria. However, due to the complexity of obtaining human host cells, many models rely on the usage of host cells derived from animals or cell lines, which does not accurately mimic the situation in the patient. Furthermore, due to the severe disbalance in host and pathogen RNA content, it is difficult to obtain sufficient sequencing depth for the infecting pathogen. Here, we present an optimized method to perform dual RNA-sequencing on human monocyte-derived macrophages (hMDMs) infected with Mycobacterium avium (M. avium) . It is likely that, with slight modifications in multiplicity of infection (MOI) to account for differences in virulence, this methodology will be applicable for other difficult-to-lyse intracellular mycobacteria.