RNA-seq of transcripts isolated from growth medium after individual growth of E. coli
For extraction of extracellular (secreted) RNAs, cultures were centrifugated for 15 min at 3000 RCF, followed by supernatant filtration using a sterile syringe and two simultaneously mounted 0.22 mkm filters, followed by purification of short RNA fraction using Qiagen miRNeasy Serum/Plasma kit (Qiagen, Germany). Libraries for sequencing were prepared using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, USA) according to the protocol of the manufacturer with modifications. MicroRNA samples were 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.