GSM6043292: RNaseE-.E.coli_sequence; Escherichia coli str. K-12 substr. MG1655; ncRNA-Seq
Total RNA from bacterial strains was extracted using the hot phenol procedure or the RiboPure™ Bacteria Kit (Invitrogen, ON, Canada, Cat. No. AM1925) following the manufacturer's recommendations. OMV-derived RNAs were isolated with RiboPure™ Bacteria Kit or RNAzol RT (Sigma, MO, USA, Cat. No. R4533) reagents following the manufacturer's recommendations. RNAzol RT kit allowed selective isolation (enrichment) of small RNAs (<200 nt). All RNA samples were subjected to treatment with DNase-I when applicable, quantified with the NanoDrop™ 2000 Spectrophotometer (Thermo Scientific™, Cat. No. ND-2000) and saved at -80°C before downstream applications. For each biological condition, an RNA sample was prepared by pooling equivalent amounts of total RNA isolated from 2 or 3 biological replicate samples. Total RNA was shipped on dry ice to the sequencing platform of Arraystar Inc. (Rockville, MD, USA). Total RNA of each sample was used to prepare the sRNA sequencing library which included the following steps: 1) 3' adapter ligation with T4 RNA ligase 2 (truncated); 2) 5' adapter ligation with T4 RNA ligase; 3) cDNA synthesis with RT primer; 4) PCR amplification; 5) extraction and purification of ~130-150 bp PCR amplified fragments from the PAGE gel. After the completed libraries were quantified with Agilent 2100 Bioanalyzer, the DNA fragments in the libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ and finally sequenced for 51 cycles on Illumina NextSeq according to the manufacturer's instruction.