Doruk, T.; Sarigoz, O.; Avican, K.

RNA sequencing has transformed our understanding of host-microbe interactions, yet in vivo profiling of bacterial pathogenesis remains fundamentally limited by the dominance of host RNA. Traditional rRNA depletion strategies fail to eliminate the vast background of host messenger and non-coding RNAs, resulting in poor pathogen coverage and necessitating cost-prohibitive deep sequencing. Here, we present a sample-derived, cDNA-guided broad host RNA depletion methodology that selectively eliminates host transcripts directly from complex infected tissues. By utilizing reverse-transcribed host RNA to guide RNase H-mediated cleavage of host RNA:cDNA duplexes, this approach enriches bacterial transcripts over 14-fold without perturbing the physiological composition of the pathogen transcriptome. Crucially, our method preserves bacterial rRNA, establishing it as a highly valuable in vivo biomarker for quantifying microbial replication rates, viability, and entry into persister states. We demonstrate that this targeted depletion achieves fully saturated bacterial gene detection at a fraction of the traditional sequencing depth, fundamentally altering the economic and computational realities of in vivo transcriptomics.