A Rapid and Cost-Effective Microcentrifuge- Based Method to Enrich for Bacterial Transcripts from Infected Eukaryotic Host Cells. Klebsiella pneumoniae strain ATCC BAA-2146

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes brute force methods inadequate for effective gene-level analysis of the pathogen. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA are eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of ~2000-3600 gene transcripts not observed in untreated control samples. The current protocol is streamlined over previous implementations of the pathogen enrichment technique, allowing 12 samples to be processed in parallel with only 1.5 hours of additional hands-on time beyond a standard RNAseq library prep.