NextSeq 500 paired end sequencing; RIL-seq experiment of identification of sRNA-target interactions in Escherichia coli K12 MG1655 arcZ::Cm strain with WT ArcZ,or a mutant with a single substitution, arcZ C81G, and a mutant with three substitutions, arcZ C81T T82G G83A plasmids
RIL-seq experiment of identification of sRNA-target interactions in Escherichia coli K12 MG1655 arcZ::Cm strain with WT ArcZ,or a mutant with a single substitution, arcZ C81G, and a mutant with three substitutions, arcZ C81T T82G G83A plasmids
E .coli Strains MG1655 z1 arcZ::Cm Hfq-flag with parcZ, pmut_C81G or p-mut_C81T_T82G_G83A and  were grown overnight in LB at 37 C with shaking (200 r.p.m.), diluted 100-fold in fresh LB, and re-grown with shaking at 37 C to stationary phase (OD600 = 1) The frozen pellet was thawed twice in a 37°C block and refrozen in liquid nitrogen. Next, the pellet was re-suspended to homogenization with warm TriReagent (1 ml) and incubated for 5 min at room temperature. Two hundred microliters of chloroform were added and the tube content was mixed by inversing the tube several times. The tube was incubated for 10 min at room temperature and centrifuged for 10 min at 4°C, 14,000 rpm. The upper phase was collected and transferred into a new Eppendorf tube. For RNA precipitation, 500 µl isopropanol was added and the tube content mixed by tube inversion. The tube was incubated for 10 min at room temperature and was centrifuged for 15 min at 4°C, 14,000 rpm. The supernatant was discarded and the pellet kept. The pellet was washed twice with 1 ml of fresh 75% (vol/vol) Ethanol (addition of Ethanol, centrifugation for 5 min at 4°C, 14,000 rpm and extraction of the supernatant) and dried for 10-15 min at room temperature. The pellet was re-suspended in 300μl nuclease free water and stored at -20°C. The experiments were performed in triplicates for each strain. RNA-seq libraries were constructed according to the RNAtag-Seq protocol (Shishkin et al., 2015) with several modifications to enable capturing of short RNA fragments: (1). After RNA fragmentation, barcoded adapter ligation and pooling, the RNA was cleaned up by RNA Clean and Concentrator 5 kit (Zymo) to maintain all RNA fragments >17 nt. (2) In all RNA cleanup steps using the RNAClean XP Beads (Beckman Coulter) and in the first strand cDNA cleanup using AMPure XP beads, 1.5 volumes of Isopropanol were added.
Experimental Factor: genetic modification
Mut ArcZ C81G