title: RNA-Seq of intracellular RNAs: E.coli in monoculture
design: Bacteria were grown in 15 ml tightly closed Hungate tubes in LB medium (pH=7.0) with 1 mg/l resazurin. Prior to inoculation, LB medium was reversibly treated with 5M NaOH, as proposed by Pavankumar and co-authors(2012, doi: 10.2144/000113819). Intracellular RNAs were isolated with TRIzol RNA extraction reagent (Invitrogen, USA) and enriched for short RNAs using mirVana miRNA Isolation Kit (Ambion, USA). Library for sequencing was prepared using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, USA) according to the protocol of the manufacturer with modifications. MicroRNA sample was subjected to adapter ligation and reverse transcription, then cleaned using Monarch PCR & DNA Cleanup Kit (New England Biolabs, USA) and fractionated in 6% polyacrylamide gel with subsequent staining with ethidium bromide. Pieces of gel containing 90-123 bp DNA fragments were cut out, minced and eluted overnight in Elution Buffer from NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1) (New England Biolabs). Then ammonium acetate (final concentration of 0.3 M) and linear acrylamide solutions were added to DNA-containing supernatant, followed by 2-hour precipitation with absolute ethanol at -80 C and centrifugation at 15000 RCF (-4 C). The pellet was washed with 80% ethanol, dried at RT for 5-10 minutes, dissolved in TE buffer from NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1) and amplified according to the protocol for Ion Total RNA-Seq Kit v2. The sample was then purified using Nucleic Acid Binding Beads from Ion Total RNA-Seq Kit v2, dissolved in pre-warmed (37 C) nuclease-free water, assessed for concentration using Qubit 3, diluted to 100 pM and used for emulsion PCR on Ion OneTouch2 System with Ion PGM Hi-Q View OT2 Kit (Thermo Fisher Scientific) and sequencing on Ion PGM (Thermo Fisher Scientific) analyzer.