Purelink rna kit

Following synchronization of cells by serum shock as described above, cells were washed with phosphate-buffered saline and returned to starvation conditions. Cells were harvested with the first time point (T = 0) taken prior to serum shock, and every 4 hours thereafter for 48 or 60 hours. Cells were harvested from separate dishes at each time point to avoid longitudinal effects/trends. Total RNA was extracted via TRIzol Reagent (Gibco) according to the manufacturer’s instructions. Briefly, 1 mL TRIzol Reagent was added to lyse the cells. Cell lysates were incubated at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes. After addition of 200 µL chloroform per 1 mL TRIzol, samples were shaken vigorously by hand for 15 seconds and incubated at room temperature for 3 minutes. Samples were then centrifuged at 12 000 × g for 15 minutes at 4°C to separate the RNA-containing, upper aqueous phase, from the lower chloroform phase. The RNA samples were further purified via PureLink RNA kit (Ambion) according to the manufacturer’s instructions. Total RNA concentration was determined via Nanodrop UV/Vis (Thermo Fisher Scientific). Overall, 1 µg of total RNA was reverse-transcribed to cDNA using 50 µM random hexamers, 40 U/µL RNaseOut, 10 mM dNTPs, and 200 U/µL SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific).

Total RNA was isolated using PureLink RNA kit (Ambion) followed by DNase I (RNase-free, Ambion) treatment. First strand cDNA synthesis was carried out using M-MuLV reverse transcriptase and random primer mix (NEB) under following conditions: 25 °C for 5 min, 42 °C for 1 h, 65 °C for 20 min. PCR amplification of cDNA samples was set with AccuStart II PCR SuperMix (Quanta BioSciences) (Pryzhkova and Jordan, 2020 (link)). Primers and PCR conditions are listed in Table S5.