A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

These experiments were designed to quantify depletion of rRNA sequencing reads from bacterial RNA-seq libraries and verify that mRNA sequencing reads were not altered. Specifically, we tested an rRNA depletion method using custom-designed biotinylated oligonucleotides and compared these results to undepleted (total RNA) libraries and libraries made with the previously-available Ribo-Zero kit (Illumina). This study consists of two sets of experiments. First, pairs of rRNA depleted libraries and total RNA libraries were prepared from the same RNA extractions to quantify depletion of rRNA by our custom biotinylated oligos. These experiments included RNA from three bacterial species. The second experiment compared the quantification of change in gene expression following treatment of E. coli with rifampicin and chloramphenicol using libraries prepared using Ribo-Zero (Illumina) or our custom biotinylated oligos. The same RNA extractions were split and treated with Ribo-Zero or our custom kit.