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.
Pcr cdna barcoding kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The PCR cDNA Barcoding Kit is a laboratory equipment product that enables the generation of barcoded cDNA libraries from RNA samples. The kit provides the necessary reagents and protocols to perform reverse transcription and PCR amplification with the addition of sample-specific barcode sequences.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 4 fluorometer, by Thermo Fisher Scientific (4 mentions)
Flowcell priming kit, by Oxford Nanopore (4 mentions)
High sensitivity rna screen tape system, by Agilent Technologies (4 mentions)
Agilent d1000 sreentape assay, by Agilent Technologies (3 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
R9.4 flow cell, by Oxford Nanopore (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Rna 6000 pico, by Agilent Technologies (1 mentions)
Qubit 1 dsdna hs assay, by Thermo Fisher Scientific (1 mentions)
Qubit broad range rna kit, by Thermo Fisher Scientific (1 mentions)
Rnaclean xp beads, by Beckman Coulter (1 mentions)
Agilent d1000 screentape assay, by Agilent Technologies (1 mentions)
Genup total rna kit, by Biotechrabbit (1 mentions)
6 protocols using pcr cdna barcoding kit
1
Direct cDNA and PCR-cDNA Sequencing
2
Nanopore Sequencing of RNA
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Quality of isolated RNA was assessed using the High Sensitivity RNA Screen Tape System (Agilent Technologies, Santa Clara, CA). Reverse transcriptase and multiplexing of the samples were performed with the PCR cDNA Barcoding Kit (SQK-PCB109, Oxford Nanopore Technologies, Oxford, United Kingdom) using 50 ng total RNA. Quantity of amplified cDNA was determined with the Qubit™ 4 Fluorometer (Invitrogen, Carlsbad, CA) and the range of fragment size was examined using the Agilent D1000 SreenTape assay (Agilent Technologies). The Flowcell (FLO-MIN106) was prepared with the FlowCell Priming Kit (EXP FLP002, Oxford Nanopore Technologies) and equal amounts of barcoded cDNA was loaded. Sequencing was carried out with a MinION (MN33710) using the MinKNOW software (v.21.02.1) over a period of 72 h.
3
Nanopore Sequencing of RNA
4
Nanopore Sequencing of Cellular and EV RNA
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The purified cellular and EV RNA samples were utilized for preparation of sequencing libraries using the PCR-cDNA Barcoding Kit (Oxford Nanopore Technologies, SQK-PCB109), as per the manufacturer’s guidelines, with some modifications. Briefly, cellular and EV samples were used for the preparation of cDNA starting with 50 ng of total RNA, followed by PCR amplification of each of the six libraries using a unique barcode per sample. The samples were subjected to 14 cycles of denaturation, annealing, and extension, and the products were purified using RNAClean XP beads (Beckman-Coulter, A66514), which can be used for both RNA and DNA purification. The purification of PCR products was conducted using the methods outlined by Oxford Nanopore. The bead-bound libraries were then eluted with 12 µL of Elution Buffer (EB) and quantified using the Qubit RNA Broad Range (BR) kit (Thermo Fisher, Q10211), as per the manufacturer’s recommendations. The samples were further analysed with Bioanalyzer to assess the library quality.
5
Nanopore Sequencing of Transcriptome
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RNAseq experiments were performed as previously described42 (link). Briefly, quality of isolated RNA was determined using the High Sensitivity RNA Screen Tape System (Agilent Technologies, Santa Clara, CA, USA). All samples showed a RIN value > 7.7. Library preparation, including reverse transcription and multiplexing of the samples was performed with the PCR cDNA Barcoding Kit (Oxford Nanopore Technologies, Oxford, United Kingdom) using 50 ng total RNA. Quantity of amplified cDNA was measured with the Qubit 4 Fluorometer (Invitrogen, Carlsbad, CA, USA) and the fragment size was examined using the Agilent D1000 ScreenTape assay (Agilent Technologies). The SpotON flow cell (R9.4.1, FLO-MIN106D) was prepared with the Flow Cell Priming Kit (EXP FLP002, Oxford Nanopore Technologies) and equal amounts of barcoded cDNA were loaded to a total of ~ 100 fmol. Sequencing was carried out with a MinION device (MN33710) using the MinKNOW software (v.21.02.1) over a period of 72 h.
6
Nanopore RNA Sequencing Protocol
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Total RNA was isolated by using the GenUP Total RNA Kit (Biotechrabbit, Hennigsdorf, Germany). The High Sensitivity RNA Screen Tape System (Agilent Technologies, Santa Clara, CA) was used to assess the quality of isolated RNA. 50 ng of total RNA was reverse transcribed and samples were barcoded with the PCR cDNA Barcoding Kit (SQK-PCB109, Oxford Nanopore Technologies, Oxford, United Kingdom). Quantity of amplified cDNA was then determined with the Qubit TM 4 Fluorometer (Invitrogen, Carlsbad, CA), and the range of fragment size was examined using the Agilent D1000 SreenTape assay (Agilent Technologies). The FlowCell Priming Kit (EXP FLP002, Oxford Nanopore Technologies) was used to prime the Flowcell (FLO-MIN106), and an equal amount of barcoded cDNA was loaded. Sequencing was carried out with a MinION (MN33710) using the MinKNOW software (v.21.02.1) over a period of 72 h.
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