The mixed RNA from each of the 18 samples was pooled in an equal quantity to obtain 1 ug RNA as required for the Oxford Nanopore library preparation. SuperScript IV First-Strand Synthesis System (Invitrogen) was used for full-length mRNA reverse transcription. According to the Oxford Nanopore recommended protocol, barcodes (Oxford Nanopore Technologies, ONT) were added to the RNA pool during cDNA amplification. The pooled cDNA was further end-repaired and dA-tailed using NEBNext Ultra End repair/dA-tailing module (NEB) and adaptor ligation using the T4 DNA ligase (NEB). Oxford Nanopore Technologies adapters were ligated to cDNA in a reaction containing adapter mix AMX1D (ONT) and Blunt/TA Ligase Master Mix (NEB). Libraries were purified by using Agencourt AMPure XP beads and eluted by Adapter Bead Binding buffer (ONT) and Elution Buffer (ONT). Libraries were then mixed with Fuel mix and Running buffer provided by ONT. Then, the final cDNA libraries were sequenced on one FLO-MIN109 flowcell and run on the PromethION platform.
Amx1d
Manufactured by Oxford Nanopore
The AMX1D is a label-free, real-time biosensing device developed by Oxford Nanopore. It utilizes nanopore technology to detect and analyze various biomolecules, including proteins, peptides, and small molecules. The core function of the AMX1D is to provide high-sensitivity detection and characterization of these analytes without the need for labeling or sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra end repair da tailing module, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Quick ligation mix, by New England Biolabs (1 mentions)
Ultra 2 end prep enzyme mix, by New England Biolabs (1 mentions)
Genejet genomic dna extraction kit, by Thermo Fisher Scientific (1 mentions)
Ampure xp magnetic beads, by Beckman Coulter (1 mentions)
2 protocols using amx1d
1
Full-Length mRNA Sequencing via Oxford Nanopore
2
Nanopore Library Preparation from Genomic DNA
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Genomic DNA was isolated from 16 million cells using the GeneJET Genomic DNA extraction kit (Thermo). Two reactions of 7 μg gDNA were then end-repaired and a-tailed using Ultra II End-prep enzyme mix (NEB) according to the manufacturer’s recommendations. Adapter mix AMX1D (Oxford Nanopore) and Quick ligation mix (NEB) were added and the reaction was incubated at room temperature for 30 min. Adapter-ligated gDNA was then purified using Ampure XP magnetic beads (Beckman Coulter) at a bead-to-sample ratio of 0.5:1. Before elution, the beads were washed twice with ABB buffer (Oxford Nanopore) then incubated with Nanopore elution buffer (Oxford Nanopore) for 15 min at 37 °C. The finished library was added to running buffer (RBF) and library loading beads (LLB) (Oxford Nanopore) then loaded onto a flow cell.
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