Protocol for isolating intracellular Salmonella RNA from primary murine macrophages

Isolation of intracellular bacterial RNA from infected cells remains challenging, limiting studies of bacterial transcription during host cell infection. Here, we present a protocol for isolating intracellular Salmonella enterica RNA from primary murine macrophages for transcriptomic analysis. We describe steps for macrophage infection, selective host cell lysis, bacterial recovery, and RNA purification for sequencing. This protocol uses standard laboratory equipment, is completed in 3 days, and can be adapted to other bacteria that replicate within macrophages. For complete details on the use and execution of this protocol, please refer to Massicotte et al.¹.