Information
This kit is highly recommended for users who:
- Want to multiplex RNA samples to reduce price per sample.
- Would like to remove PCR bias
- Are exploring attributes of native RNA such as modified bases
Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.
The Direct RNA Sequencing Kit features:
| Feature | Property |
|---|
| Preparation time | ~370 minutes |
| Input requirement | 450 fmol of poly(A)+ enriched RNA, or 450 fmol of poly(A)-tailed IVT transcript RNA in 5 μl |
| PCR required | No |
| Fragmentation | No |
| Read length | RNA length |
| Associated protocols | • Direct RNA sequencing - barcoding (SQK-DRB004.24) |
| Pack size | 6 sequencing library preparations |
| Stability | This kit is stable during shipping for 7 days at temperatures up to 25ºC. Upon receipt, please store the kit under the recommended conditions specified on the kit label.
The warranty for this product is 3 months from receipt by the customer. |
The Direct RNA Barcoding Kit 24 (SQK-DRB004.24) is used to barcode, prepare and sequence multiple native RNA without conversion to cDNA. Inputs include enriched poly(A)+ enriched RNA samples or poly(A)-tailed IVT transcript samples.
Please note: This kit is optimised for multiplexing 4–8 poly(A)+ enriched RNA samples, or 12-24 poly(A)-tailed IVT transcript samples. To ensure you are multiplexing the correct number of samples, please consider the sequencing output you require per sample for your analysis.
This kit includes increased sequencing output and improved modal raw read accuracy on our RNA flow cell (FLO-MIN004RA and FLO-PRO004RA). The flow cell priming reagents are included as part of the sequencing kit and are specific to our RNA flow cells. Other features include fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.
Further considerations:
In applications where Direct RNA sequencing’s key advantages (detection of modified bases and reduced bias) are not important, users may consider one of Oxford Nanopore’s cDNA kits (SQK-PCS114 and SQK-PCB114.24), which currently provide the highest output per run.
Shipping and logistics:
Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.
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.
Workflow
The Direct RNA Barcoding Kit 24 (SQK-DRB004.24) is used to perform multiplex sequencing of native RNA, starting from poly(A)+ enriched RNA or from poly(A)-tailed IVT transcripts. Reverse transcription barcodes are ligated to your samples, before pooling samples together. The barcoded samples are then reverse transcribed to perform first-strand synthesis, providing stability to the native RNA strand. A USER digestion reaction is performed to digest the overhangs from the excess Reverse Transcription Barcodes, preventing barcode cross-contamination. Sequencing adapters are then attached to the RNA-cDNA hybrid for sequencing.
What's in the box
The Direct RNA Barcoding Kit 24 contains 24 unique barcodes and sufficient reagents for up to 6 library preparation reactions.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|
| RNA Barcode Adapter | RBA | Green | 1 | 45 |
| RNA Elution Buffer | REB | Black | 1 | 300 |
| Displacement Strand | DS | Orange | 1 | 10 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| EDTA | EDTA | Blue | 1 | 700 |
| RNA Flush Tether | RFT | Purple | 1 | 200 |
| S Fragment Buffer | SFB | Clear bottle | 1 | 13,000 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
| Reverse Transcription Barcode plate | RTB01-24 | - | 2 plates, 3 sets of barcodes per plate | 5 µl per well |
3rd party materials
Consumables
- T3 DNA Ligase (NEB, M0317)
- Induro® Reverse Transcriptase and 5x Induro® RT Reaction Buffer (NEB, M0681)
- Murine RNase Inhibitor (NEB, M0314)
- USER (Uracil-Specific Excision Reagent) Enzyme (NEB, M5505L)
- Agencourt RNAClean XP beads (Beckman Coulter®, A63987)
- 10 mM dNTP solution (e.g. NEB N0447)
- 5M NaCl, RNase-free (ThermoFisher, 10609823 or equivalent)
- NEBNext High Input Poly(A) mRNA Isolation Module (NEB, E3370)
- Nuclease-free water (e.g. Thermo Scientific, AM9937)
- Qubit™ RNA HS Assay Kit (ThermoFisher, Q32852)
- Qubit™ dsDNA HS Assay Kit (ThermoFisher, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- 1.5 ml Eppendorf DNA LoBind tubes
- PCR plate seals
Equipment:
- Vortex mixer
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic separation rack, suitable for 1.5 ml Eppendorf tubes
- Eppendorf 5424 centrifuge (or equivalent)
- Ice bucket with ice
- Timer
- Thermal cycler
- Qubit™ fluorometer (or equivalent for QC check)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
Note: The above are 3rd party reagents required for the standard prep. For other workflows using this kit, extra 3rd party reagents may be required.
Compatibility
The Direct RNA Barcoding Kit 24 can be used together with:
Kits
- Direct RNA Auxiliary Vials (EXP-DRA004)
- RNA Control Expansion (EXP-RCS001)
Flow cells
- FLO-MIN004RA
- FLO-PRO004RA
Devices
- MinION
- GridION
- PromethION
