title: GSM8239477: HEK293T cells, rep3; Homo sapiens; ncRNA-Seq
library_name: GSM8239477
library_construction_protocol: Total RNA was harvested using TRIzol (ThermoFisher Scientific). Total RNA was deacylated in 100 mM CHES-NaOH (pH 9.0) at 37 °C for 30 min and purified by ethanol precipitation. Then deacylated RNAs were incubated with the recombinant AlkB WT (1.5 μM) and L118V D135S (0.1 μM) in the presence of 1 mM α-ketoglutarate, 2 mM L-ascorbic acid, 50 μM (NH4)2Fe(SO4)2 in 50 mM HEPES-KOH (pH 8.1) at 37 °C for 1 h, followed by phenol/chloroform extraction and ethanol precipitation. RNAs were further incubated with 10 U of T4 polynucleotide kinase (New England BioLabs) at 37 °C for 1 h, and purified using RNA Clean & Concentrator-5. Then samples were separated using 10% 7M urea gel and the bands corresponding to tRNAs were excised and subjected to gel-extraction. tRNA samples were ligated with adenylated adaptors using T4 RNA Ligase 2, truncated KQ (New England BioLabs), followed by gel-purification. Then samples were reverse-transcribed using TGIRT-III (InGex) and subjected to alkaline hydrolysis. Then samples were circularized using CircLigase II (Lucigen) and purified, followed by the PCR amplification using Phusion Polymerase (ThermoFisher Scientific). Sequencing was performed using NextSeq 500 Sequencer and NextSeq 500/550 Mid-Output v2 Kit (Illumina).