GSM6588635: Target_293T_ACTB_EEF2_RPL5_RPS6_mock_BEU; Homo sapiens; OTHER
Total RNA was extracted from cells using TRIzol reagent (Life Technologies). The selection of polyA(+) RNA was performed using Dynabeads™ Oligo(dT)25 (Invitrogen). RNA-seq of transfected HEK293T cells were performed with TRACE-seq method. For CeU-seq and Target-seq, the RNA pellet was dissolved in 20 μl 5 mM EDTA, incubated at 80°C for 5 minutes, and quickly chilled on ice. Next, 10 μl denatured RNA was transferred into 100 μl in BEU buffer (50 mM Bicine, pH 8.5, 4 mM EDTA, 7 M urea) with 0.2 M CMC (Sigma, Cat#C106402, 95%) as the CMC+ sample, or into 100 μl BEU buffer as the CMC- control sample. Both CMC+ and CMC- samples were then incubated at 37 °C for 20 min, followed by purification with ethanol precipitation. The RNA pellets were then dissolved in 50 μl Na2CO3 buffer (50 mM Na2CO3 pH 10.4, 2 mM EDTA) and incubated at 37 °C for 6 h, followed by ethanol precipitation.