This kit is highly recommended for users who:

• would like to identify and quantify full-length transcripts
• are interested in differential gene expression
• want to characterise and quantify isoforms, splice variants and fusion transcripts
• wish to avoid bias and artefacts introduced through PCR

The Direct cDNA Sequencing Kit features:

Description

The Direct cDNA Sequencing Kit is used to prepare cDNA for nanopore sequencing without using PCR. The kit recommends an input of 100 ng poly-A⁺RNA.

Taking full-length poly-A⁺ RNA, complementary strand synthesis and strand switching are performed using kit-supplied oligonucleotides. The RNA template is then digested, and the second (complementary) strand is synthesised. Sequencing adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the dscDNA.

Further considerations:

A Native Barcoding Expansion pack (EXP-NBD104) can be used to multiplex up to 12 different RNA samples into a single flow cell.

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

Taking full-length messenger RNA, the complementary strand is synthesised using a kit-supplied oligo adapter. The RNA is then degraded, and the second (complementary) strand synthesised. Adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the cDNA, which introduce the components needed for the cDNA strands to enter the pore. One strand of the duplex is sequenced at a time, producing 1D reads.

Example protocol using Direct cDNA Sequencing Kit - for illustration purposes only

Example protocol using Direct cDNA Sequencing Kit - PromethION - for illustration purposes only

Safety data sheet for Direct cDNA Sequencing Kit (SQK-DCS109)

Safety Data Sheet for Flow Cell Priming Kit

The Direct cDNA Sequencing Kit contains sufficient reagents to generate six sequencing libraries.

The Flow Cell Priming Expansion Pack is also supplied.

PCR-free multiplexing with barcoding for up to 12 samples is available with an additional purchase: Native Barcoding Expansion 1-12 (EXP-NBD104) and/or Native Barcoding Expansion 13-24 (EXP-NBD114).

Consumables

• NEBNext End repair / dA-tailing Module (E7546)
• NEB Blunt/TA Ligase Master Mix (M0367)
• 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
• Agencourt AMPure XP beads
• 10 mM dNTP solution (e.g. NEB cat # N0447)
• LongAmp Taq 2X Master Mix (e.g. NEB cat # M0287)
• Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
• RNaseOUT™, 40 U/μl (Life Technologies, cat # 10777019)
• RiboShredder (Epicentre, RS12500), or RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286)

Equipment

• Hula mixer (gentle rotator mixer)
• Magnetic rack
• Microfuge
• Vortex mixer
• Ice bucket with ice
• Timer
• Thermal cycler
• P1000 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P10 pipette and tips
• P2 pipette and tips
• Pre-chilled freezer block at -20° C for 200 µl tubes (e.g. Eppendorf cat # 022510509)

Optional Equipment

• Agilent Bioanalyzer (or equivalent)
• Qubit fluorometer (or equivalent for QC check)
• Eppendorf 5424 centrifuge (or equivalent)

The Direct cDNA Sequencing Kit can be used together with:

Kits

• Native Barcoding Expansion 1-12 (EXP-NBD104)
• Native Barcoding Expansion 13-24 (EXP-NBD114)
• Flow Cell Wash Kit (EXP-WSH003)

Flow cells

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

Devices

• MinION Mk 1B
• GridION X5
• PromethION