This kit is recommended for users who:

• wish to multiplex samples to reduce price per sample
• have a limited amount of input material
• want to optimise their sequencing experiment for throughput
• would like to identify and quantify full-length transcripts
• are interested in differential gene expression or differential transcript usage
• want to characterise and quantify isoforms, splice variants and fusion transcripts

The PCR-cDNA Barcoding Kit features:

Description

Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.

The PCR-cDNA Barcoding Kit is used to prepare cDNA for nanopore sequencing for up to 12 samples, from an input of as low as 1 ng poly-A⁺ RNA. Users who do not have poly-A⁺ enriched RNA can use 50 ng of total RNA but additional optimisation may be required.

Taking full-length poly-A⁺ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together, and Rapid Sequencing Adapters are added to the pooled mix.

Shipping and logistics

Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8° C, and -20° C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer.

Taking full-length poly-A⁺ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The kit contains 12 primer pairs which are used to generate and then amplify double-stranded cDNA by PCR amplification: each primer pair contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together, and Rapid Sequencing Adapters are added to the pooled mix.

Data analysis

Data from a PCR-cDNA sequencing experiment can be analysed using a number of bioinformatics tools and pipelines:

• DESeq2 for differential gene expression profiling
• pinfish for annotation of gene transcripts and novel gene isoform discovery
• pipeline-transcriptome-de for performing differential transcript usage (DTU) and differential gene expression (DGE) analyses

Oxford Nanopore Technologies offers easy-to-follow bioinformatics tutorials, which take the user step-by-step through each analysis pipeline. These tutorials are available in the Nanopore Community.

Example protocol using the PCR-cDNA Barcoding Kit (SQK-PCB109) – for illustration purposes only

Example protocol using the PCR-cDNA Barcoding Kit (SQK-PCB109) - PromethION – for illustration purposes only

Safety Data Sheet for the PCR-cDNA Barcoding Kit (SQK-PCB109)

Safety Data Sheet for the Flow Cell Priming Kit (EXP-FLP002)

The PCR-cDNA Barcoding Kit contains sufficient reagents to generate six sequencing libraries.

The Flow Cell Priming Expansion Pack is also supplied.

Consumables

• Agencourt AMPure XP beads
• 1.5 ml Eppendorf DNA LoBind tubes
• 0.2 ml thin-walled PCR tubes
• Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
• Freshly prepared 70% ethanol in nuclease-free water
• 10 mM dNTP solution (e.g. NEB N0447)
• LongAmp Taq 2X Master Mix (e.g. NEB M0287)
• Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
• RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
• Exonuclease I (NEB, M0293)

Equipment

• Hula mixer (gentle rotator mixer)
• Magnetic separator, suitable for 1.5 ml Eppendorf tubes
• Microfuge
• Vortex mixer
• Thermal cycler
• P1000 pipette and tips
• P200 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P10 pipette and tips
• P2 pipette and tips
• Ice bucket with ice
• Timer
• Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf 022510509)
• Qubit fluorometer (or equivalent for QC check)

The PCR-cDNA Barcoding Kit can be used together with:

Kits

• Flow Cell Wash Kit (EXP-WSH003)

Flow cells

• FLO-MINSP6
• FLO-MIN106D
• FLO-PRO002

Devices

• MinION Mk 1B
• GridION Mk1
• PromethION