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Cell culture, treatments, and RNA purification

SK Svetlana Kalmykova
MK Marina Kalinina
SD Stepan Denisov
AM Alexey Mironov
DS Dmitry Skvortsov
RG Roderic Guigó
DP Dmitri Pervouchine
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A549 cell line (kindly gifted by Dr. Ilya Terenin, Moscow State University) was maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 0.05 mg/ml streptomycin (all products from Thermo Fisher Scientific) at 37 °C in 5% CO2. Cell line authentication was confirmed using short tandem repeat analysis. The cell line was tested for the absence of mycoplasma using MycoReport kit (EuroGene). For α-amanitin (Sigma) treatments, 1 and 2 μg/mL of α-amanitin was added to cells at 50–70% confluency. After 24 h of treatment, cells were harvested, total RNA was isolated using PureLink RNA Mini Kit (Thermo Fisher Scientific). Poly(A)+ mRNA was purified using Dynabeads Oligo(dT) 25 (Thermo Fisher Scientific) following the manufacturer’s instructions.

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