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Notas
| Methodology:
Promastigotes of L. Infantum JPCM5 strain (MCAN/ES/98/LLM-724) were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum. Logarithmically growing cells were harvested by centrifugation, and total RNA was isolated by the NucleoSpin RNA kit following the manufacturer’s instructions (Macherey-Nagel). RNA integrity was checked in a bioanalyzer (Agilent 2100) before proceeding with cDNA synthesis.
RNA Library preparation made following the instructions and Direct RNA Sequencing Kit (SQK-RNA002, ONT) reagents. Briefly: approx. 500 ng of total RNA in 9 µL (Nuclease-free Water) was taken, mixed and span down. In a 0.2mL PCR tube, the following components were added: 3 µl NEBNext Quick Ligation Reaction Buffer (NEB B6058), 9 µL RNA, 0.5 µL RNA CS (RCS) 110 nM (Direct RNA Sequencing kit), 1 µL of RT Adapter (RTA) (Direct RNA Sequencing Kit) and 1.5 µL T4 DNA Ligase 2M U/mL(NEB M0202). The reagents were mixed, span down, and incubated 10 min at RT. Then, the reverse transcription mix was made by mixing: 9 µL Nuclease-free Water (Ambion, AM9938), 2 µL 10 mM dNTPs (Biotools, 20.031-4179), 8 µL 5x first-strand buffer (Thermo Fisher Scientific, 18080044) and 4 µL 0.1M DTT (Roche, 10708984001). This mix was added to the 0.2mL PCR tube previously prepared with the RT adapter-ligated RNA and mixed. Then, 2 µL of SuperScript III reverse transcriptase (Thermo Fisher Scientific, 18080044) was added. Using a thermal cycler (Techne, TC-3000), the mix was incubated at 50°C for 50 min, then 70°C for 10 min, and brought to 4°C; finally, the solution was transferred to a new clean 1.5mL tube. 72 µL of resuspended Agentcourt RNAClean XP beads (Beckman Coulter, A63987) were added to the reaction, mixed and incubated on a rotator mixer for 5 min at RT. The sample was span down and pelleted on a magnet (DynaMag, InvitroGen, 123.20D). Supernatant was pipetted off. Beads were washed with 150 µL of freshly prepared 70% ethanol, with two rotations of 180°, and pelleted. The supernatant was discarded by pipetting. The pellet was suspended in 20 µL nuclease-free water and incubated 5 min at RT. Then, 20 µL of eluate were pipetted into a new clean 1.5mL tube, where the following reagents were added and mixed: 8 µl NEBNext Quick Ligation Reaction Buffer (NEB B6058), 6 µL RNA Adapter (RMX) (Direct RNA Sequencing Kit), 3 µL nuclease-free water, and 3 µL T4 DNA Ligase 2M U/ml (NEB M0202). Afterwards, 16 µL of RNAClean XP beads were added to the reaction, mixed by pipetting, and incubated on a rotator mixer for 5 min at RT. The sample was span down and pelleted on a magnet, and the supernatant was discarded. Then, 150 µL of the Wash Buffer (WSB) (Direct RNA Sequencing Kit) were added to the beads, suspended and pelleted with a magnetic rack. The supernatant was pipetted off. The pellet was suspended in 21 µL of Elution Buffer (Direct RNA Sequencing Kit) and incubated 10 min at RT. The beads were pelleted on a magnet. Finally, 21 µL of this eluate, containing the RNA library, were removed and retained into a clean 1.5 mL tube, which was later used for sequencing.
Priming and Loading the NanoPore SpotON flow cell was performed by using the Direct RNA Sequencing Kit (SQK-RNA002, ONT) and the Flow Cell Priming Kit (EXP-FLP002) as indicated in the manufacturer's protocol. Briefly, the RNA Running Buffer (RRB), the Flush Buffer (FB) and the Flush Tether (FLT) were vortexed at RT. 30 µL of FLT were added to the tube of FB (1170 µL) and mixed, generating the priming mix. Around 20-30 µL of buffer was removed from the priming port for avoiding bubbles. Then, 800 µL of priming mix were added to the cell through the priming port, and incubated for 5 min. Then, 200 µL of priming mix were added to the priming port after lifting the SpotON cover. 20 µL of the RNA library, 17.5 µL of nuclease-free water, and 37.5 µL of RRB were mixed and added to the Flow Cell. The Priming port and the MinION device were closed. Sequencing was performed with MinION Mk1C (ONT) MC-114562. The flow cell ID was FAW00477, where 301 pores were found to be available. Basecalling was performed with MinKNOW (ONT) software model version rma002_70bps_hac@v3, with firmware MinION FPGA 2.4.3, ver. 23.07.12. |
