title: NextSeq 500 sequencing; RNA-Seq samples of E. coli K12 MG1655, deletion of ryhB sRNA gene, 2,2'-Dipyridy
design: RNA-Seq samples of E. coli K12 MG1655, deletion of ryhB sRNA gene, 2,2'-Dipyridy
library_construction_protocol: centrifuged for 5 min at 4°C, 4500g, resuspended in 50 µl TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) and mixed with 5 ul of  9 mg/ml lysozyme prior to freezing the sample at liquid nitrogen and storing it at -80°C. Bacterial strains used in this study are listed in Table S1. The gene expression levels of two strains were compared: (1) a strain in which the sRNA is induced for a short time, from the endogenous chromosomal locus (in the study of RyhB). (2) A control strain in which the sRNA is not induced, a deletion strain of the sRNA gene, a ryhB deletion strain. Bacteria were routinely grown at 37°C in LB rich medium. In all experiments, single colonies were suspended in liquid medium and grown over night. The next day the cultures were diluted 1:100 in fresh medium and grown to an OD600 of 0.5. Where indicated, the iron chelator 2,2'-Dipyridy (200 µM final; DIP) was added to strain (1) or (2) the culture for the last 30 minutes of growth. Bacterial strains used in this study are listed in Table S1. The gene expression levels of two strains were compared: (1) a strain in which the sRNA is induced for a short time, from the endogenous chromosomal locus (in the study of RyhB). (2) A control strain in which the sRNA is not induced, a deletion strain of the sRNA gene, a ryhB deletion strain. Bacteria were routinely grown at 37°C in LB rich medium. In all experiments, single colonies were suspended in liquid medium and grown over night. The next day the cultures were diluted 1:100 in fresh medium and grown to an OD600 of 0.5. Where indicated, the iron chelator 2,2'-Dipyridy (200 µM final; DIP) was added to strain (1) or (2) the culture for the last 30 minutes of growth. Frozen samples were subjected to two cycles of thawing at 37°C and refreezing in liquid nitrogen. Next, the samples were re-suspended thoroughly to homogenization with 1 mL TriReagent (pre-warmed 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 l isopropanol were 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, 5 min at 4°C) and removal of the supernatant. Pellets were air dried by at room temperature, and then re-suspended in 300 L nuclease free water and stored at -20°C. The RNA concentration was measured using Nanodrop (ThermoFisher Scientific). The experiments were performed in triplicates for each strain. RNA-seq libraries were constructed according to the RNAtag-Seq protocol {Shishkin, 2015 #973} with several modifications, to enable capturing of short RNA fragments {Melamed, 2018 #1252}. The libraries were single-end sequenced with read length of 92 bases on NexstSeq500 Sequencer (Illumina).