title: Microcolony-seq applied on a clinical urinary tract infection
library_construction_protocol: 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 37°C. 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. The frozen samples were subjected to two cycles of thawing at 37°C and refreezing in liquid nitrogen. Next, the samples were resuspended thoroughly to homogenization with 1 mL TriReagent (prewarmed to room temperature) and incubated for 5 min at room temperature. Two hundred microliters of chloroform were added and the tubes content was mixed by inversing the tubes for 15 s. The samples were incubated for 10 min at room temperature, centrifuged (17,000g, 10 min at 4°C) and the upper phase was collected and transferred into new Eppendorf tubes. For RNA precipitation, 500 μl isopropanol was added, the tube contents were mixed thoroughly by inversion of the tubes and incubated for 10 min at room temperature. The tubes were centrifuged (17,000g,15 min at 4°C) and the supernatant was discarded. The pellets were washed twice by addition of 1 mL of freshly made 75% (vol/vol) ethanol, followed by centrifugation (17,000g for 5 min at 4°C) and removal of the supernatant. Pellets were dried by leaving the tubes open for 15 min at room temperature, and then re-suspended in 300 μL nuclease free water and stored at −20°C. The RNA concentration was measured using Nanodrop (ThermoFisher Scientific). rRNA depletion was done by using the DIY rRNA depletion method (Culviner et al., 2020), using the rRNA sequence probe set designed for E. coli K-12. RNA-seq libraries were constructed based on the RNAtag-Seq protocol (Shishkin et al., 2015) with several modifications (Melamed et al., 2018). Since the rRNA depletion step was done in advance, after the fragmentation step the first ligation was carried out.