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. 2018 Aug 2;8:11606. doi: 10.1038/s41598-018-29707-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Validation of the RNA sequencing approach using digital PCR (dPCR) with six candidate markers. Control:treated ratios (C:T ratios) determined by RNA sequencing (red) were validated against C:T ratios measured by dPCR (gray). The dPCR C:T ratios were normalized using ribosomal RNA (rRNA) by dividing the C:T ratio of the candidate marker by the C:T ratio of 16S rRNA. This normalization step is not required for sequencing data because the values are normalized by sequencing depth (see Methods). Markers were validated using two susceptible (S1 and S2) and two resistant (R1 and R2) isolates at 15 min of ciprofloxacin exposure.