**2. RNA isolation and bacterial RNA/cDNA enrichment**

The widely used scheme of an intracellular bacterial transcriptome analysis is illustrated by Fig. 1.

Fig. 1. Stages of intracellular transcriptome analysis (left) and main approaches used to enrich the cDNA library by bacterial transcripts (right).

The methods of RNA isolation and enrichment are described in details in our recent review (Skvortsov & Azhikina, 2010). Here we would like to emphasize that until the present time, practically all ways of preparing cDNA libraries have lead to the further microarray hybridization analysis. Hybridization-based microarray techniques are widely used in transcriptome studies, but development of novel high-throughput DNA sequencing methods (reviewed in (Shendure & Ji, 2008)) obviously demonstrated such microarrays limitations as high background levels owing to cross-hybridization, problems of rare transcript detection, and expression quantification (Shendure, 2008).

We proposed a new method for the evaluation of sequences of bacterial pathogens specifically transcribed in infected tissues (Azhikina et al., 2010). It is based upon the coincidence cloning approach that allows isolation of representative bacterial cDNA pools from infected organs. Co-denaturation and co-renaturation of the excess of bacterial genomic DNA with the cDNA transcribed from total RNA of the infected tissue enabled selective isolation of the bacterial cDNA fraction from the sample, and a single round of coincidence cloning resulted in >1000-fold enrichment of bacterial transcripts. The enriched cDNA library is suitable for high-throughput sequencing analysis, which is less biased and more reliable than other methods including microarrays.
