title: GSM8252120: HCT116_KO_rep2; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted using TRIzol reagent following manufacturer's instructions. tRNAs were isolated from total RNA by ion exchange chromatography. The RNA samples were brought to a final concentration of 20 mM HEPES-KOH (pH 7.5) and loaded onto a Bio-Rad EconoFit Macro-Prep DEAE columns (BioRad, 12009274) pre-equilibrated sequentially with the following buffers: 6 column volumes (CV) of low salt buffer (200 mM NaCl, 8 mM MgCl2, 20 mM HEPES-KOH pH 7.5), followed by 15 CV of high salt buffer (450 mM NaCl, 16 mM MgCl2, 20 mM HEPES-KOH pH 7.5), then 15 CV of low salt buffer, and finally, 15 CV of binding buffer (100 mM NaCl, 20 mM HEPES-KOH pH 7.5). After loading the RNA, the column was washed with 10 CV of binding buffer and eluted with a step gradient of 200 mM NaCl, 8 mM MgCl2, 20 mM HEPES-KOH to 500 mM NaCl, 16 mM MgCl2, 20 mM HEPES-KOH. A final elution step was conducted with 1 M NaCl. After elution, the collected fractions were analyzed by on urea-PAGE, and the fractions containing pure tRNA were pooled and subsequently recovered by precipitation with 1/10 volume of 3M sodium acetate (pH 5.2), 20 ug/ml Glycogen and 1.5 volume of isopropanol. The tRNA samples purified from total RNA were prepared into libraries following the Nano-tRNAseq protocol developed by Lucas et al. (Nature Biotechnology, 2024, 42, 72–86). Initially, the tRNAs were deacylated in 100 mM Tris-HCl (pH 9.0) at 37°C for 30 minutes. After recovery using the NEB RNA Cleanup kit, the tRNAs were ligated to pre-annealed 5' and 3' splint adaptors (5' RNA splint adapter: 5'-rCrCrUrArArGrArGrCrArArGrArArGrArArGrCrCrUrGrGrN, 3' splint adapter: 5'-Phos-rGrGrCrUrUrCrUrUrCrUrUrGrCrUrCrUrUrArGrGrArArArArArArArArArAAAA-3') with T4 RNA 2 Ligase followed by purification on AMPure RNAClean XP beads. The splint adaptor-ligated tRNAs were reverse transcribed using SuperScript IV Reverse Transcriptase at 60 °C for 1 hour followed by 85 °C for 5 minutes to linearize the tRNAs. The linearized tRNAs were cleaned up with AMPure RNAClean XP beads and then prepared into libraries following the standard protocol of the Direct RNA Sequencing Kit (SQK-RNA002) before being sequenced on a MinION flow cell (FLO-MIN-106).