NextSeq 500 sequencing; RNA-seq data of Escheirchia coli K12 MG1655 of overexpression of GcvB, MicA, ArcZ, RyhB or CyaR sRNAs
RNA-seq data of Escheirchia coli K12 MG1655 of overexpression of GcvB, MicA, ArcZ, RyhB or CyaR sRNAs
For GcvB induction, the E. coli K-12 MG1655 Z1 gcvB::Cm, pZA12-gcvB and the E. coli K-12 MG1655 Z1 gcvB::Cm, pTP-011 strains were used. For MicA overexpression the E. coli K-12 MG1655lacIq pBRplac, pEF21-Hfq and the E. coli K-12 MG1655lacIq pMicA, pEF21-Hfq strains were used. For ArcZ induction the E. coli K-12 MG1655 Z1 arcZ::Cm, pZE12-ArcZ and the E. coli K-12 MG1655 Z1 arcZ::Cm, pJV300 strains were used. For RyhB overexpression the E. coli K-12 MG1655 ryhB::Cm and the E. coli K-12 MG1655  were used. For CyaR induction the E. coli K-12 MG1655 Z1 cyaR::Cm, pZE12-CyaR and the E. coli K-12 MG1655 Z1 cyaR::Cm, pJV300 strains were used. All E. coli strains used in this study were grown overnight in Luria Bertani (LB) medium at 37 °C with shaking (200 r.p.m.), diluted 100-fold in fresh LB medium, and re-grown with shaking at 37 °C to exponential phase, for GcvB (OD600 = 0.3) and for RyhB (OD600 = 0.5), or to stationary phase, for ArcZ WT, ArcZ M1, ArcZ M2 and CyaR (OD600 = 1.0) and for MicA (grown for 6 hr). For induction of ArcZ and GcvB, IPTG was added (1mM, 20 min). MicA was constitutively expressed and further induced at the end of growth with IPTG (1mM, 20 min). For induction of RyhB, the iron chelator 2,2'-Dipyridyl was added (200 μM, 30 min). After the growth of the bacteria to the relevant condition, the culture was centrifuged at 4°C, 5000 rpm and resuspended in 50 µl TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). Lysosyme was added to 0.5 mg/ml and the pellet was frozen at liquid nitrogen and stored at -80°C. 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
delta cyaR+pJV300