AbstractDual transcriptional profiling of host and bacteria during infection is challenging due to the low abundance of bacterial mRNA. We report Pathogen Hybrid Capture (PatH-Cap), a method to enrich for bacterial mRNA and deplete bacterial rRNA simultaneously from dual RNA-seq libraries using transcriptome-specific probes. By addressing both the differential RNA content of the host relative to the infecting bacterium and the overwhelming abundance of uninformative structural RNAs (rRNA, tRNA) of both species in a single step, this approach enables analysis of very low-input RNA samples. By sequencing libraries before (pre-PatH-Cap) and after (post-PatH-Cap) enrichment, we achieve dual transcriptional profiling of host and bacteria, respectively, from the same sample. Importantly, enrichment preserves relative transcript abundance and increases the number of unique bacterial transcripts per gene in post-PatH-Cap libraries compared to pre-PatH-Cap libraries at the same sequencing depth, thereby decreasing the sequencing depth required to fully capture the transcriptional profile of the infecting bacteria. We demonstrate that PatH-Cap enables the study of low-input samples including single eukaryotic cells infected by 1–3 Pseudomonas aeruginosa bacteria and paired host-pathogen temporal gene expression analysis of Mycobacterium tuberculosis infecting macrophages. PatH-Cap can be applied to the study of a range of pathogens and microbial species, and more generally, to lowly-abundant species in mixed populations.