Direct cdna sequencing kit

Manufactured by Oxford Nanopore

The Direct cDNA Sequencing Kit by Oxford Nanopore is a laboratory tool designed for direct cDNA sequencing. It enables the direct sequencing of cDNA molecules without the need for prior amplification or fragmentation.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Kapa pure beads, by Roche (1 mentions) Primescript double strand cdna synthesis kit, by Takara Bio (1 mentions) Kapa hyper prep kit, by Roche (1 mentions) Minion platform, by Oxford Nanopore (1 mentions) Native barcoding expansion kit, by Oxford Nanopore (1 mentions) Flongle flow cell, by Oxford Nanopore (1 mentions) Tks gflex dna polymerase, by Takara Bio (1 mentions) Minion mk1b device, by Oxford Nanopore (1 mentions) Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions) Gridion, by Oxford Nanopore (1 mentions) Turbo dnase, by Thermo Fisher Scientific (1 mentions) Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions) Qiashredder column, by Qiagen (1 mentions) Rneasy mini column, by Qiagen (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Longamp taq master mix, by New England Biolabs (1 mentions) Nebnext ultra 2 end repair da tailing module, by New England Biolabs (1 mentions) Rnase cocktail, by Thermo Fisher Scientific (1 mentions)

5 protocols using direct cdna sequencing kit

Transient overexpression and silencing of NbFKPPIase

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N. benthamiana leaves after being infiltrated with agrobacteria containing the NbFKPPIase transient overexpression or silencing construct were collected for RNA extraction. A total amount of 1.5 µg RNA per sample was used for transcriptome analysis. Double‐stranded cDNA was synthesized using the PrimeScript Double‐Strand cDNA Synthesis Kit (Takara) following the manufacturer's recommendations. DNA was cleaned up with KAPA Pure Beads (KAPABiosystem; Roche) followed by end repair and A‐tailing reaction with the KAPA HyperPrep Kit (KAPABiosystems). DNA was cleaned up for adaptor ligation and subjected to sequencing with a Direct cDNA Sequencing Kit (Oxford Nanopore Technology). A total amount of 100 pmol of cDNA was loaded on Nanopore R9.4 flow cells and sequenced using the MinION platform (Oxford Nanopore Technology).

Chang H., Lee C., Chang C, & Jan F. (2022). FKBP‐type peptidyl‐prolyl cis‐trans isomerase interacts with the movement protein of tomato leaf curl New Delhi virus and impacts viral replication in Nicotiana benthamiana. Molecular Plant Pathology, 23(4), 561-575.

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Influenza Virus Genomic Sequencing

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Nucleotide sequences of the open reading frames of all viral gene segments were determined by nanopore sequencing using the MinION Mk1b device (Oxford Nanopore Technologies, Oxford, UK), as previously described [13 (link)]. Briefly, the eight-segmented influenza viral genes were individually amplified by conventional PCR using Tks Gflex DNA Polymerase and KOD One PCR Master Mix -Blue- (TaKaRa Bio Inc.) with gene segment-specific primer sets [15 (link)] using the cDNAs synthesized above. The PCR amplicons were cleaned up and adaptor-ligated using a Direct cDNA Sequencing Kit (Oxford Nanopore Technologies) with a Native Barcoding Expansion Kit (Oxford Nanopore Technologies), and sequenced with Flongle flow cells (Oxford Nanopore Technologies) using MinION control software (Oxford Nanopore Technologies). The consensus sequences for each gene segment were generated using Geneious Prime v.2021.1.1 (Biomatters Ltd., Auckland, New Zealand). The nucleotide sequences were deposited in the Global Initiative on Sharing Avian Influenza Data (GISAID) database (http://platform.gisaid.org/) on 2 September 2022 (Table 1).

Okuya K., Khalil A.M., Esaki M., Kojima I., Nishi N., Koyamada D., Matsui T., Yoshida Y, & Ozawa M. (2022). Genetic Characterization of Avian Influenza Viruses Isolated from the Izumi Plain, Japan in 2019/20 Winter Season. Pathogens, 11(9), 1013.

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Nanopore Sequencing of Phanerochaete Mitogenomes

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The mycelia of P. noxius KPN91 were cultured on the PDA agar plates at 25 °C for about 3–5 days. Total RNA was extracted from mycelia homogenized in liquid nitrogen, following with TRIzol Reagent (catalog no. 15596018, Thermo Fisher Scientific, Inc.). Ploy-A enriched RNA (200 ng) was purified from total RNA by using Dynabeads mRNA purification kit (catalog no. 61006, Thermo Fisher Scientific, Inc.) and was used to conduct cDNA library preparation with Direct cDNA Sequencing kit (SQK-DCS108; Oxford Nanopore Technologies). The reads were sequenced with FLO-MIN106 flow cell on a GridION, Oxford Nanopore and then basecalled by dogfish v0.9.3. Illumina RNA-seq reads of four species in this study (P. noxius, P. lamaensis, Po. pini, and Con. sulphurascens) were downloaded and aligned to corresponding assemblies using STAR (v2.6.0a; Dobin et al. 2013 (link)). Nanopore cDNA reads (P. noxius) were aligned using minimap2 (v2.6; Li 2018 (link)). Only reads aligned to mitogenome were retained for further analysis.

Lee H.H., Ke H.M., Lin C.Y., Lee T.J., Chung C.L, & Tsai I.J. (2019). Evidence of Extensive Intraspecific Noncoding Reshuffling in a 169-kb Mitochondrial Genome of a Basidiomycetous Fungus. Genome Biology and Evolution, 11(10), 2774-2788.

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Poly(A) RNA sequencing from cell lysates

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Cell lysates were homogenized with QIAshredder columns (QIAGEN #79654) and total RNA was extracted with RNeasy mini columns (QIAGEN #74104). RNA was DNase treated using TURBO DNase (ThermoFisher Scientific #AM2238). RNA quality was verified on a 2100 Bioanalyzer (Agilent Technologies) and polyadenylated RNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490L). Libraries were generated using 100 ng of poly(A) + RNA and Oxford Nanopore Technologies’ Direct cDNA Sequencing Kit (ONT SQK-DCS109), with each sample barcoded with the Native Barcoding Expansion kits (ONT EXP-NBD104 and ONT EXP-NBD114), according to the manufacturer’s specifications. Barcoded samples were pooled, loaded on a MinION sequencer on R9 Flow Cells (ONT FLO-MIN106D), and sequenced using the MinKNOW software v19. The long-read RNA-sequencing data generated in this study have been deposited in the GEO database under accession code GSE184081.

Varga B.V., Faiz M., Pivonkova H., Khelifi G., Yang H., Gao S., Linderoth E., Zhen M., Karadottir R.T., Hussein S.M, & Nagy A. (2022). Signal requirement for cortical potential of transplantable human neuroepithelial stem cells. Nature Communications, 13, 2844.

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RNA Extraction and cDNA Synthesis for Nanopore Sequencing

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RNA was extracted and purified from approximately 25–30 mg of homogenised tissue with an RNeasy Kit (Qiagen – 74104) supplementing the RLT buffer with 1% (v/v) ß-mercaptoethanol (Sigma-Aldrich – M3148). The purity and concentration of RNA was assessed spectrophotometrically using a NanoDrop (Thermo Scientific, model ND-1000). For each sample, 100 ng of extracted RNA was converted to cDNA using RNaseOUT (Invitrogen – 10777019), Maxima H Minus Reverse Transcriptase (Thermo Scientific – 15259496), primers from a Direct cDNA Sequencing Kit (Oxford Nanopore – SQK-DCS109), RNase Cocktail (Invitrogen – AM2286), LongAmp Taq Master Mix (New England Biolabs – M0287), NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs – E7546), and AMPure XP magnetic beads (Beckman Coulter – A63881). cDNA was then assessed spectrophotometrically, as above, for use in Nanopore sequencing.

Elcombe C.S., Monteiro A., Ghasemzadeh-Hasankolaei M., Evans N.P, & Bellingham M. (2021). Morphological and transcriptomic alterations in neonatal lamb testes following developmental exposure to low-level environmental chemical mixture.. Environmental toxicology and pharmacology, 86, 103670.

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