GSM6123621: HEK293T FUSKO 2; Homo sapiens; OTHER
Total RNA was isolated using the Quick-RNA MiniPrep Kit (Zymo Research, R1055) according to the manufacturer's protocol. 250 ng of RNA from each replicate was used for hydrazine treatment for 30-60 min on ice. To stop the reaction, ethanol precipitation with 0.3 M NaOAc, pH 5.2 and Glycoblue was performed, and incubated at −80°C for 30 min, followed by centrifugation. The collected pellet was washed with 80% ethanol and resuspended in 1 M aniline, pH 4.5, followed by incubation in the dark for 15 min at 60°C and then ethanol precipitation. Dephosphorylation of RNA at the 3' end was performed. Library preparation was carried out using the NEBNext Small RNA Library kit (NEB, UK), following the manufacturer's protocol. ncRNA-Seq: HydraPsiSeq, a quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage.