RNA-seq of E. coli K-12 MG1655 grown in M9 medium containing size-selected mid-length oligonucleotides
Overnight cultures of E. coli K12 MG1655 were equalized to OD 600 = 0,5. Cells were inoculated v/v 1:1000 and grown in 30 ml M9 mineral medium with shaking at 37 for 12 or 24 hours. Samples from the first set were supplemented with 50 nM of oligonucleotide 5GCCAAGGC(G)-3 and harvested after 24 hours of incubation. The second experiment was performed with 25 nM of the oligonucleotide, and cells were harvested after 12 hours of incubation. Cells from the culture were sedimented by centrifugation at 5000 rpm (4), rinsed with PBS and resuspended in TRIzol reagent (Invitrogen, Carlsbad, CA, USA).  The sample was then subjected to three freeze-thaw cycles in liquid nitrogen and at 37 before proceeding with the standard TRIzol protocol.  Total RNA fraction samples were treated with DNAse I (New England Biolabs, USA) according to the manufacturer's protocol.  Afterwards, RNA fractions were purified using the CleanRNA Standard kit (Evrogen, Russia). To prepare cDNA libraries, 1 g of each RNA sample was polyadenylated with Poly(A) polymerase (TransGen Biothech Co, China). Polyadenylated RNA was cleaned up with AMPure XP beads (Beckman Coulter, Brea, CA, USA). Its concentration was measured with a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Singapore) using the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Eugene, OR, USA). cDNA synthesis was performed using the Mint kit (Evrogen, Russia). After 24-26 amplification cycles, prior to reaction saturation, libraries of double-stranded cDNA fragments were fractionated on a 5% TBE polyacrylamide gel alongside a Quick-Load 50bp DNA Ladder (New England Biolabs, Ipswich, MA, USA). cDNAs of the desired size ( 500 bp) were excised and purified using ammonium acetate. The final cDNA concentration was measured using the Qubit 3.0 fluorometer (Thermo Fisher Scientific, Singapore) and QuDye dsDNA HS (Lumiprobe, Russia).