GSM7109009: OCI-AML2 cells, TRMT5 sgRNA KO biological rep2; Homo sapiens; OTHER
Mitochondria were isolated using a MACS antibody based mitochondria extraction kit (Miltenyi Biotech) to obtain high quality and intact mitochondria for subsequent RNA isolation. Subsequently, small and large RNAs were isolated using the miRNeasy Micro Kit (Qiagen). In order to perform NanotRNA-seq on tRNAs, small RNA fractions isolated from KO and scrambled were deacylated with the following protocol; 10 µL nuclease-free water and deacylated in 95 µL 100 mM Tris-HCl (pH 9.0) at 37°C for 30 min. Deacylated tRNAs were recovered using Zymo RNA Clean and Concentrator-5 kit (Zymo, R1016), following the manufacturer's instructions. tRNA libraries were prepared using NanotRNA-seq library protocol with some protocol alterations described here. The 5' and 3' splint adapters were mix at a 1:1 molar ratio in a solution of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl and 1 µL RNasin® Ribonuclease Inhibitor (Promega, N251A) to a final concentration of 1 uM. The mix was heated to 75°C for 15 s and cooled to 25°C at a rate of 0.1°C/s to anneal splint adapters. 200 ng deacylated tRNAs were ligated to the pre-annealed splint adapters at a molar ratio of 1.2:1 (with median length of 90 nucleotides. Deacylated tRNAs were ligated to the pre-annealed 5' and 3' splint adapters at a molar ratio of 1.2:1 (assuming an average tRNA length of 90 nt). The ligation was carried out at room temperature for 2 hours in 25 µL with 20% PEG PEG 8000 (NEB, B10048), 1X T4 RNA Ligase 2 Buffer (NEB, B0239S), 2 µL 6 mg/mL recombinant E. coli T4 RNA 2 Ligase (made in-house) and 0.5 µL RNasin® Ribonuclease Inhibitor. Ligation reaction was then cleaned up with 2X volume of RNAClean XP beads (Beckman, A63987) following manufacturer's instructions. The RNA concentration was determined using Qubit™ RNA High Sensitivity (HS) assay kit (Thermo, Q32852). At least 50 ng of 5' and 3' ligated tRNAs were ligated to the RTA adapter (ONT, SQK-RNA002)  at a molar ratio of 1:2 in the presence of 1X Quick Ligation Reaction buffer (NEB, B6058S), 1.5 μL T4 DNA Ligase (NEB, M0202M, 2,000,000 units/mL) and 0.5 µL RNasin® Ribonuclease Inhibitor in 15 µL reaction at room temperature for 10 minutes. Consequently, the reaction mix was added to a reverse transcription mix of 11 µL nuclease-free water, 2 µL 10 mM dNTPs (NEB, N0447S), 8 µL 5X Maxima H Minus Reverse Transcriptase Buffer, and 2 µL Maxima H Minus Reverse Transcriptase (Life Technologies, EP0751). Reverse transcription mix was then mixed well and incubated at 60°C for 1 hour, 85°C for 5 minutes and then brought to 4°C. The linearized tRNAs were cleaned up using 2X RNAClean XP beads following manufacturer's instructions. Then, 6 ul ONT RMX sequencing adapters (ONT, SQK-RNA002) were ligated at room temperature for 10 minutes in a total reaction volume of 40 µL with 1X Quick Ligation Reaction buffer, 3 μL T4 DNA Ligase. The ligation mix was cleaned up with 2X volume of RNAClean XP beads  following manufacturer's instructions with a few adjustments. The sample was washed twice with 150 μL WSB (Wash Buffer), in which the pellet was resuspended by flicking the tube. The sample was eluted in 20 μL ELB (Elution Buffer) and incubated for 10 minutes at room temperature on a Hula mixer. The final library was prepared by adding 17.5 μL of nuclease-free water and 37.5 μL of vortexed RRB and kept on ice until loading. The MinION flow cell (FLO-MIN-106) was quality checked, primed, and loaded following the standard ONT SQK-RNA002 protocol.