Guscn

Lysis buffer was prepared to a final concentration of 5 M Guanidium thiocyanate, by dissolving 120 g guanidinium thiocyanate (GuSCN) (Sigma Aldrich) in 100 mℓ of 0.1 M hydroxymethyl amino methane-hydrochloric acid (Tris-HCl) (pH 6.4) (Sigma Aldrich) in a 500 mℓ Schott bottle. The bottle was heated to 60 °C to dissolve the GuSCN. After heating, 22 mℓ of a 0.2 M ethylenediamine tetra-acetate (EDTA) (pH 8.0) with 2.6 mℓ triton X-100 (Sigma Aldrich) solution was added to the suspension. The suspension was mixed and dispensed into 50 mℓ Eppendorf tubes and 0.5 mℓ of celite suspension was added to remove any contaminating DNA from the buffer. The final solution was left to stand for at least 1 h at room temperature with sporadic mixing. The celite was pelleted by centrifugation at 3000 rpm for 10 min (NeoFuge-15R, Heal Force, Vacutec®) and the supernatant was transferred into sterile 50 mℓ Eppendorf tubes wrapped in aluminium foil (stable 3 weeks at room temperature).

For each 10 cm dish cell, added 100 μl of 6 M GuSCN (Sigma, 368975) and lysed cells with vigorous manual shaking for 1 min. Then, cell lysate was added 12 μl of 500 mM EDTA (Invitrogen™, 15575020), 60 μl of 10× PBS (Invitrogen^™, AM9625), and water to a final volume of 600 μl. Each sample was passed through a 25 or 26 G needle about 20 times to further break the insoluble material. Proteinase K (PK) (Thermo Scientific^™, EO0492) was added to a final concentration of 1 mg/ml, and PK treatment was performed at 37 °C for 1 h on a shaker at 1000–1200 × g. After PK digestion, 60 μl of 3 M sodium acetate (pH 5.3) (Invitrogen^™, AM9740), 600 μl of water-saturated phenol (pH 6.6) (Invitrogen^™, AM9712), and 1 volume pure isopropanol were added to precipitate total nucleic acids by spinning at 17,000 × g for 20 min at 4 °C. After twice washing using 70% ethanol, total nucleic acids were resuspended in 300 μl of nuclease-free water. For 100 μg of TNA samples, 50 units of TURBO^™ DNase (Invitrogen^™, AM2239) were added to remove DNA at 37 °C for 20 min. Then added 20 μl of 3 M sodium acetate, an equal volume of water-saturated phenol, two-volume of pure isopropanol to precipitate RNA sample by spinning 20 min at 12,000 × g at 4 °C.

Tris HCl, GuSCN, MgCl2, HCl, NaOH, CH3COOH, NaHCO3, NaOAc, 2-mercaptoethanol, octanoic acid, isopropanol, and ethanol were acquired from Sigma-Aldrich (St. Louis, MO, USA). NaCl, phosphate-buffered saline (PBS), and Dextran blue 2000 kDa (DEXB) were obtained from Applichem (Darmstadt, Germany), and Merck KGaA (Darmstadt, Germany), respectively. Na2EDTA was obtained from Fluka (Geneva, Switzerland), and PEG20000 was obtained from Loba Chemie (Mumbai, India). EDTA spray-coated vacutainers were obtained from Fisher Scientific (Waltham, MA, USA). Glycogen, the RiboLockRNAse inhibitor, the MMLV reaction buffer, and MMLV reverse transcriptase were acquired from Fermentas (Vilnius, Lithuania). Primers and probes for reverse transcription and TaqMan qPCR (Table 1) and dNTPs were synthesized at the organic synthesis Laboratory of ICBFM SB RAS (Novosibirsk, Russia), and the 10× PCR buffer and Taq DNA polymerase were obtained from BiolabMix (Novosibirsk, Russia). Cel-miR-39-3p was synthesized in the Laboratory of Medicinal Chemistry (ICBFM SB RAS, Novosibirsk, Russia).