Microcolony-seq applied on a clinical urinary tract infection

Urine was obtained from a patient with a urinary tract infection and frozen in 15% glycerol at -80C. For the Microcolony-seq experiment a sample from the frozen urine was serially diluted four times 10-fold and plated on an LB agar plates and incubated at 37C. After 8 hours of incubated microcolonies were visible. Twenty microcolonies were picked and subjected to the Microcolony-seq pipeline and four additional microcolonies were mixed and separated to four Eppendorf tubes and served as the technical replicates of the experiment. RNA was extracted with 1 mL TriReagent (Sigma-Aldrich) per sample. RNA quality was assessed by Nanodrop and by Bioanalyzer using the Agilent RNA 6000 Pico Kit (5067-1513). rRNA depletion was done by using the DIY rRNA depletion method, using the rRNA sequence probe set designed for E. coli K-12. RNA-seq libraries were constructed based on the RNAtag-Seq protocol with several modifications. The rRNA depletion step was done first following by the fragmentation step in the RNAtag-Seq protocol. Then the first ligation was carried out and the rest of the RNAtag-Seq protocol was followed. Libraries were single-end sequenced using the Nextseq500 Sequencer (Illumina).