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R9.4 flow cell

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The R9.4 flow cell is a core consumable used with Oxford Nanopore's sequencing platforms. It serves as the primary component for DNA or RNA sample loading and analysis. The flow cell contains nanopores that enable the direct, real-time detection and identification of individual molecules passing through them.

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Lab products found in correlation

Ampure xp bead, by Beckman Coulter (8 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (8 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions) Qubit 3.0 fluorimeter, by Thermo Fisher Scientific (5 mentions) Ligation sequencing kit, by Oxford Nanopore (5 mentions) Ligation sequencing kit 1d, by Oxford Nanopore (3 mentions) Native barcoding kit, by Oxford Nanopore (3 mentions) Ligation sequencing kit sqk lsk109, by Oxford Nanopore (3 mentions) Sqk rna001 nanopore drs kit, by Oxford Nanopore (2 mentions) Cdna pcr sequencing sqk pcs108 kit, by Oxford Nanopore (2 mentions) Rnase free zirconium oxide beads, by Next Advance (2 mentions) Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (2 mentions) Qiazol lysis reagent, by Qiagen (2 mentions) Qiazol, by Qiagen (2 mentions) Bullet blender, by Next Advance (2 mentions) Guppy basecaller, by Oxford Nanopore (2 mentions) Q5 hot start high fidelity dna polymerase, by New England Biolabs (2 mentions) Native barcoding expansion kits 1 12 and 13 24, by Oxford Nanopore (2 mentions) Qubit double stranded dna, by Thermo Fisher Scientific (2 mentions) Genomic dna buffers kit, by Qiagen (2 mentions)

26 protocols using r9.4 flow cell

1

Direct RNA Sequencing with Calibration

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Direct RNA-sequencing libraries were generated from the isolated poly(A) RNA, spiked with 0.25 µl of a synthetic Enolase 2 (ENO2)-derived calibration strand (a 1.3 kb synthetic poly(A)+ RNA, Oxford Nanopore Technologies Ltd.) and sequenced on one of two MinION MkIb with R9.4 flow cells (Oxford Nanopore Technologies Ltd.) and an 18-h runtime. All protocol steps are described in Garalde et al.22 (link). Following sequencing, basecalling was performed using Albacore 1.2.1 [-f FLO-MIN106 -k SQK-RNA001 -r -n 0 -o fastq, fast5]. Only reads present in the “pass” folder were used in subsequent analyses.
Depledge D.P., Srinivas K.P., Sadaoka T., Bready D., Mori Y., Placantonakis D.G., Mohr I, & Wilson A.C. (2019). Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen. Nature Communications, 10, 754.
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2

Direct cDNA and PCR-cDNA Sequencing

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Libraries for direct cDNA and PCR-cDNA sequencing were prepared following the instructions in the direct cDNA sequencing with native barcoding protocol (SQK-DCS109 with EXP-NBD104) and the PCR-cDNA Barcoding Kit (SQK-PCB109) from Oxford Nanopore Technologies with minor modifications. Briefly, the VN Primer was replaced with a custom 3′ cDNA RT primer (5′-ACTTGCCTGTCGCTCTATCTTCATTGATGGTGCCTACAG-3′, 2 µM). The size of the input RNA and the strand-switched (direct cDNA) or PCR-amplified cDNA (PCR-cDNA) was assessed via a Bioanalyzer (Agilent) run using the RNA 6000 Pico and the High Sensitivity DNA Kit (Agilent), respectively. After RT, samples used for PCR-cDNA sequencing were amplified for 12 cycles at an elongation time of 5 min. During library preparation, the quantity and quality of the samples were tested using standard spectroscopic measurements (Nanodrop One) and using the Qubit 1× dsDNA HS Assay and Qubit RNA HS Assay Kit (Thermo Fisher Scientific). Finally, samples were pooled in equimolar ratios, adapter-ligated or attached following the respective protocols, loaded onto R9.4 flow cells (Oxford Nanopore Technologies) and sequenced on a MK1C device for 48 h.
Grünberger F., Jüttner M., Knüppel R., Ferreira-Cerca S, & Grohmann D. (2023). Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea. RNA, 29(8), 1255-1273.
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3

Nanopore Direct RNA Sequencing of Arabidopsis

Cited 1 time
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Total RNA was isolated from Col-0, fpa-8 and 35S::FPA:YFP seedlings as described above. mRNA was isolated and Nanopore DRS libraries prepared (using the SQK-RNA001 Nanopore DRS Kit; Oxford Nanopore Technologies) as previously described (Parker et al., 2020 (link)). Libraries were loaded onto R9.4 flow cells (Oxford Nanopore Technologies) and sequenced using a 48 hr runtime. Four biological replicates were performed for each genotype.
Parker M.T., Knop K., Zacharaki V., Sherwood A.V., Tomé D., Yu X., Martin P.G., Beynon J., Michaels S.D., Barton G.J, & Simpson G.G. (2021). Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA. eLife, 10, e65537.
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4

Nanopore Sequencing of Amplicons

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We sequenced amplicons from 4 UR and 15 INS samples using the Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) according to the protocol described in Quick et al. [20 (link)]. Amplicons were barcoded using the Native Barcoding Kit EXP-NBD103 (Oxford Nanopore Technologies, Oxford, UK) and pooled in equimolar fashion. Sequencing libraries were prepared using the 1D Genomic DNA Sequencing kit SQK-LSK108 (Oxford Nanopore Technologies, Oxford, UK). We used AMPure XP beads (Beckman Coulter, Brea, CA, USA) for all purification steps performed as part of library preparation. Prepared libraries were sequenced on R9.4 flowcells (Oxford Nanopore Technologies, Oxford, UK) at the INS in Bogotá and at the Fred Hutchinson Cancer Research Center in Seattle.
Black A., Moncla L.H., Laiton-Donato K., Potter B., Pardo L., Rico A., Tovar C., Rojas D.P., Longini I.M., Halloran M.E., Peláez-Carvajal D., Ramírez J.D., Mercado-Reyes M, & Bedford T. (2019). Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia. BMC Infectious Diseases, 19, 963.
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5

Betulaceae Genome Sequencing Protocol

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DNA extraction and sequencing was carried out as part of an ongoing C. avellana genome sequencing project. High molecular weight DNA was extracted from young leaf buds using a CTAB method optimized for Betulaceae [68 (link)]. Whole genome shotgun libraries were prepared using TruSeq kits and selected for an insert size of 600–800 nt. Paired-end sequencing was carried out on a Illumina HiSeq4000 and reads were deposited in the European Nucleotide Archive (Project accession: PRJEB31933). NanoPore sequencing reads were also obtained for the same cv Tombul genome project. NanoPore sequencing was carried out on the MinION platform using R9.4 flowcells and Ligation Sequencing Kit 1D, according to the manufacturer’s protocols (Oxford NanoPore Technologies, Oxford, UK).
Kahraman K, & Lucas S.J. (2019). Comparison of different annotation tools for characterization of the complete chloroplast genome of Corylus avellana cv Tombul. BMC Genomics, 20, 874.
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6

Long-read Sequencing of ESBL Isolates

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We conducted long-read WGS on a subset of isolates by using the MinION Platform (Oxford Nanopore Technologies, https://nanoporetech.com). We prepared libraries by using the Rapid Barcoding SQK-RBK004 Kit and sequenced them by using R9.4 Flowcells (both from Oxford Nanopore Technologies). All blaSHV-2 isolates were selected for long-read sequencing because this gene was commonly detected in humans (n = 12) and animals/meat (n = 15). If an ESBL variant was observed >5 times in 1 source (humans or animals/meat), we selected a convenience sample of 3 isolates from that source for long-read sequencing (blaCTX-M-55 and blaCTX-M-65 in humans and blaCTX-M-1 in animals/meat). If the ESBL enzyme was observed <5 times in 1 source, we selected 1 isolate for long-read sequencing (blaSHV-2, blaCTX-M-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, and blaCTX-M-15 in humans and blaCTX-M-55 in animals/meat).
Bharat A., Mataseje L., Parmley E.J., Avery B.P., Cox G., Carson C.A., Irwin R.J., Deckert A.E., Daignault D., Alexander D.C., Allen V., El Bailey S., Bekal S., German G.J., Haldane D., Hoang L., Chui L., Minion J., Zahariadis G., Reid-Smith R.J, & Mulvey M.R. (2022). One Health Genomic Analysis of Extended-Spectrum β-Lactamase‒Producing Salmonella enterica, Canada, 2012‒2016. Emerging Infectious Diseases, 28(7), 1410-1420.
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7

Direct RNA Nanopore Sequencing Protocol

Cited 1 time
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Libraries for direct RNA nanopore sequencing were prepared from poly(A)-tailed RNAs according to the SQK-RNA002 Kit protocol (Oxford Nanopore, Version: DRS_9080_v2_revS_14Aug2019) with minor modifications. Briefly, Agencourt AMPure XP magnetic beads (Beckman Coulter) in combination with 1 µL of RiboGuard RNase Inhibitor (Lucigen) were used instead of the recommended Agencourt RNAclean XP beads to purify samples after enzymatic reactions. Also, the RTA adapter was replaced by custom adapters described in https://github.com/Psy-Fer/deeplexicon to barcode the samples (Smith et al. 2020 (link)). RNA was linearized using Maxima RT H Minus enzyme (Thermo Fisher Scientific, #EP0751) and incubated for 30 min at 60°C followed by a heat inactivation step of 5 min at 85°C. After RT reactions, cDNA was quantified using the Qubit DNA HS Assay Kit (Thermo Fisher Scientific), and equimolar amounts of DNA were used for ligation of the RNA Adapter (RMX) in a single tube. Subsequent reactions were performed according to the protocols recommended by Oxford Nanopore Technologies. Finally, libraries were loaded onto R9.4 flow cells (Oxford Nanopore Technologies) and sequenced on a MinION device for 48 h. Direct RNA sequencing was performed for two biological replicates of wild-type and KsgA-deletion samples.
Grünberger F., Jüttner M., Knüppel R., Ferreira-Cerca S, & Grohmann D. (2023). Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea. RNA, 29(8), 1255-1273.
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8

Nanopore Direct RNA Sequencing

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Total RNA was isolated from Col-0, fpa-8 and 35S::FPA:YFP seedlings as described above.
mRNA was isolated and Nanopore DRS libraries prepared (using the SQK-RNA001 Nanopore DRS Kit; Oxford Nanopore Technologies) as previously described (Parker et al., 2020) . Libraries were loaded onto R9.4 flow cells (Oxford Nanopore Technologies) and sequenced using a 48-h runtime. Four biological replicates were performed for each genotype.
Parker M.T., Knop K., Zacharaki V., Sherwood A.V., Tomé D.F.A., Yu X., Martin P.G., Beynon J., Michaels S.D., Barton G.J., & Simpson G.G. (2020). Widespread premature transcription termination ofArabidopsis thalianaNLR genes by the spen protein FPA.
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9

Sequencing of the K. humilis Genome

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Sequencing of the K. humilis MAW1 genome was performed using a 2 × 300 PE MiSeq strategy in the DNA Sequencing and Synthesis Facility at IBB PAS. For Oxford Nanopore MinION sequencing, gDNA of K. humilis MAW1 was fragmented into 8–10 kbp fragments using a Covaris gTUBE (7200 rpm for 60 s). The gDNA was then processed with a Ligation Sequencing Kit 1D (Oxford Nanopore, SQK-LSK108). Sequencing was performed with a R9.4 flow cell (Oxford Nanopore, FLO-MIN106) in a MinION Mk1B.
Mielecki D., Detman A., Aleksandrzak-Piekarczyk T., Widomska M., Chojnacka A., Stachurska-Skrodzka A., Walczak P., Grzesiuk E, & Sikora A. (2024). Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties. Microbial Cell Factories, 23, 111.
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10

Testis tissue RNA extraction and sequencing

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Testis tissue was homogenized at 4°C in 1ml QIAzol Lysis Reagent (Qiagen, Hilden, Germany) together with RNase-Free Zirconium Oxide Beads (NextAdvance, Inc., Troy, NY, USA) during 1 min, maximum effect, in a BulletBlender (NextAdvance) and total RNA was prepared according to the QIAzol (Qiagen) protocol. Polyadenylated RNA was isolated with the Dynabeads mRNA direct purification kit (Thermo Fisher Scientific), following the kit protocol. For full-length cDNA Oxford Nanopore sequencing, the cDNA-PCR Sequencing SQK-PCS108 kit (Oxford Nanopore Technologies) was used according to the kit protocol with minor modifications. The finished cDNA library was sequenced on a MinION device using a R9.4 flow cell (Oxford Nanopore Technologies).
Edvardsen R.B., Wallerman O., Furmanek T., Kleppe L., Jern P., Wallberg A., Kjærner-Semb E., Mæhle S., Olausson S.K., Sundström E., Harboe T., Mangor-Jensen R., Møgster M., Perrichon P., Norberg B, & Rubin C.J. (2022). Heterochiasmy and the establishment of gsdf as a novel sex determining gene in Atlantic halibut. PLoS Genetics, 18(2), e1010011.
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