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Notas
| Methodology:
Promastigotes of L. braziliensis (strain M2904) 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. The original RNA concentration was 567 ng/uL.
RNA Library preparation made following the instructions and some reagents from the Direct RNA Sequencing kit (SQK-RNA002, ONT). In brief, 500 ng of total RNA (9 µL in nuclease-free water) were mixed with 3 µl of NEBNext Quick Ligation Reaction Buffer (NEB B6058), 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 of T4 DNA Ligase 2 MU/mL(NEB M0202). Afterwards, the mixture was incubated for 10 min at room temperature (RT). Then, the following components were added: 9 µL of nuclease-free water (Ambion, AM9938), 2 µL of 10 mM dNTPs (Biotools, 20.031-4179), 8 µL of 5x first-strand buffer (Thermo Fisher Scientific, 18080044), 4 µL of 0.1 M DTT (Roche, 10708984001), and 2 µL of SuperScript III reverse transcriptase (Thermo Fisher Scientific, 18080044). Using a thermal cycler (Techne, TC-3000), the mix was incubated at 50ºC for 50 minutes, then 70ºC for 10 minutes, and chilled to 4ºC. Afterwards, 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 pelleted on a magnet (DynaMag, InvitroGen, 123.20D). Supernatant was pipetted off. Beads were washed with 150 µL of freshly prepared 70% ethanol, and pelleted. The 70% ethanol was discarded by pipetting. Pellet was resuspended in 20 µL nuclease-free water and incubated 5 min RT. Then, the following reagents were added: 8 µl of NEBNext Quick Ligation Reaction Buffer (NEB B6058), 6 µL RNA Adapter (RMX, Direct RNA Sequencing Kit), 3 µL nuclease-free water, and 3 µL of T4 DNA Ligase 2 MU/ml (NEB M0202). Afterwards, 16 uL of resuspended RNAClean XP beads were added to the reaction, mixed by pipetting, and incubated on a rotator mixer for 5 min at RT. Sample was pelleted on a magnet, and the supernatant was discarded. 150 µL of the Wash Buffer (WSB, Direct RNA Sequencing Kit) were added to the beads, resuspended and pelleted with a magnetic rack. The supernatant was pipetted off. The pellet was resuspended in 21 µL of Elution Buffer (Direct RNA Sequencing Kit) and incubated for 10 min at RT. The beads were pelleted on a magnet. Finally, this eluate, containing the RNA library, were removed and retained into a clean 1.5 mL tube, which was then used for the sequencing step. 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 protocol. Briefly, the RNA Running Buffer (RRB), the Flush Buffer (FB) and the Flush Tether (FLT) were vortexed at RT. 30uL 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 was 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 FAY36834, where 1286 pores were found 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.
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