title: GSM8840259: HT29, TYW2, rep2; Homo sapiens; OTHER
library_construction_protocol: For tRNA extraction, total RNA was isolated using Trizol reagent (Invitrogen), according to manufacturer's instructions. The samples were treated with Turbo DNase (Thermo Fisher Scientific) and small RNA fraction containing tRNAs (≤200 nt) was retained using a Zymo RNA Clean and Concentrator-5 kit (Zymo Research). For deacylation, the retained fraction incubated in 50 mM Tris-HCl (pH 9.0) at 37 °C for 30 minutes and recovered using a Zymo RNA Clean and Concentrator-5 kit (Zymo Research). The tRNA profiles were confirmed using Agilent 2100 Bioanalyzer, RNA 6000 Nano assay tRNAs libraries were prepared as previously described (figure 1a) and according to nanopore direct RNA sequencing protocols with some adjustments. The libraries were prepared using either  SQK-RNA002 kit (ONT) or SQK-RNA004 kit (ONT) and all oligos used were acquired from Integrated DNA Technologies (IDT). In short, deacylated tRNA samples were ligated to an annealed adapter set that is complementary to the 3' NCCA tRNA overhang and contains short poly(A) tail, using T4 RNA ligase 2 (New England Biolabs) for 2h at room temperature Then, DNA oligonucleotides with identical sequence to ONT RTA adapters were annealed and ligated using T4 DNA ligase (New England Biolabs) for 30 minutes at room temperature. The ligated tRNA samples were converted into cDNA using Maxima H Minus Reverse Transcriptase cDNA synthesis kit (Life Technologies). After each ligation step, annealed tRNAs were cleaned and recovered using CleanNGS kit (CNGS-0050, GC biotech) according to the manufacturer protocol, measured on nanodrop and their profiles were confirmed using Agilent 2100 Bioanalyzer, Small RNA analysis (Supplementary figure S1a). Finally, the samples were ligated to either ONT RMX sequencing adapters according to SQK-RNA002 kit (ONT) protocol or to RNA Ligation Adapter (RLA) according to SQK-RNA004 kit (ONT) and recovered using CleanNGS kit (CNGS-0050, GC biotech). The library was prepared for loading according to protocol then loaded on the appropriate RNA MinION Flow Cell (RNA002 or RNA004). Simulation runs were performed on the bulk recorded data of the RNA002 flow cells
identifiers.namespace: GEO
identifiers.id: GSM8840259_r1