Fig. 1.

Schematic of EMBR-Seq. After performing an optional Terminator™ 5′-phosphate-dependent exonuclease digestion, poly-A polymerase and rRNA blocking primers (purple) are added to total bacterial RNA (mRNA in red and rRNA in gray). Blocking primers specifically bind to the 3′ end of 5S, 16S, and 23S rRNAs, resulting in the preferential addition of a poly-A tail to mRNA molecules. Next, reverse transcription is performed using (i) a poly-T primer, which has an overhang containing a sample-specific barcode (blue), 5′ Illumina adapter (yellow), and T7 promoter (green), and (ii) rRNA blocking primers to convert poly-adenylated RNA and rRNA molecules, respectively, to cDNA. The cDNA molecules are then amplified by in vitro transcription, and the amplified RNA is used to prepare Illumina libraries. As the rRNA-derived cDNA does not contain a T7 promoter, these molecules are not amplified during in vitro transcription, resulting in rRNA depletion