Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3), the RNA was split into the poly(A)+ and poly(A)− fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A)− fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A)− transcriptome from pathogen and host cells as well as the poly(A)+ fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.