R300 Zymo-Seq RiboFree Total RNA-Seq Library Kit is the fastest, easiest RNA-Seq library prep kit available to make stranded, total RNA libraries. The automation-friendly protocol minimizes hands-on time to generate sequencer-ready, stranded, indexed libraries depleted of rRNA in as little as 3.5 hours. Depletion of ribosomal RNAs (which can comprise as much as 90% of a total RNA sample) is completely integrated into the workflow, using a novel, probe-free technology that removes rRNA or any other over-abundant, repetitive transcripts from any sample type or species. RiboFree Universal Depletion is validated across biological kingdom and phyla, including human, rodent, plant, and various prokaryotes, as well as RNA from a wide range of sample types including cells, tissue, FFPE tissues, and whole blood, eliminating the need to select sample-specific modules for different sample types. Just one total RNA-Seq library prep kit to deplete them all. Because the RiboFree Universal Depletion is probe-free, researchers using the Zymo-Seq RiboFree Total RNA Library Kit will avoid transcriptome profiling bias found in other methods. High concentrations of rRNA-binding oligonucleotides used in probe-based approaches also bind to a significant number of non-ribosomal targets. As much as 20% of the 20,000 protein-coding genes in the human genome are significantly biased by these methods. In contrast, the probe-free depletion developed by Zymo Research uses only abundant pre-existing transcripts for mismatch-free enzymatic removal of rRNA or other over-abundant transcripts, resulting in minimal off-target effects as low as 1.8% in all protein-coding transcripts. Pre-mixed reagents are ready-to-use at every step, eliminating the need for manual master-mix preparation. Everything needed to make sequencing-ready libraries, including indexing primers and SPRI beads are included in the kit. It’s RNA-Seq made simple.
| Equipment Required | Thermocycler, magnet stand*, and microcentrifuge.
A complimentary magnet stand is available at online checkout for direct U.S. customers of R3000 and R3003. |
|---|---|
| Input Quality | Ensure RNA A260/A280 and A260/A230 ratios are ≥ 1.8, DNA-free, and PCR inhibitor-free for high-fidelity cDNA transcription and depletion |
| Maximum Input | 5 µg |
| Minimum Input | 100 ng |
| Processing Time | As little as 3.5 hours (RNA to indexed library) |
| Recommended Input | 500 ng |
| Sample Input Material | RNA from any species |
| Sequencing | Libraries are stranded and compatible with all Illumina® sequencing platforms. The Read 1 sequence will be antisense to the RNA molecule of origin. |
| Supplemental Info |
Protocol: R3000
Datasheet
SDS (MSDS): R3000 | R3003
Barcode Sequences Table
Sample Sheet Templates: MiSeq | NovaSeq v1.5 Chemistry
Example Bioinformatic Commands
Q1: What samples are compatible with this kit?
Purified total RNA from any organism. Please see our Multi-Organism Transcriptomics Application Note or find.
Q2: Is this kit suitable for metatranscriptomics?
This kit is not designed for metatranscriptomics, and thus the performance cannot be guaranteed for such application. In our preliminary test using total RNA extracted from our ZymoBIOMICS Microbial Community Standard (D6300), using 500 ng as input and depleting for at least 2 hours yielded libraries where remaining rRNA reads were around 20%. Therefore, for metatranscriptomic samples with low complexity (<10 microbial species/strains), this kit could be suitable, and we recommend using 500 ng of total RNA whenever possible and perform depletion for at least 2 hours. For further information, please contact tech@zymoresearch.com.
Q3: Is this kit suitable for degraded RNA with low RIN scores?
Yes, this kit works with degraded RNA with low RIN scores, such as RNA from FFPE samples, although the performance may be negatively impacted. For optimal results using degraded samples, we recommend increasing the input amount, amplifying with more PCR cycles, and adopting column cleanup using RNA Clean & Concentrator without DNase I treatment for the depletion section. For details, please refer to Appendix E in the kit protocol or contact tech@zymoresearch.com.
Q4: What if my RNA sample is contaminated with DNA?
DNA contamination will adversely impact the accuracy and sensitivity of quantitative measures of gene expression and differential gene expression analysis. Therefore, we recommend removing DNA contamination from the RNA input prior to performing the Zymo-Seq RiboFree workflow. For extracted RNA, we recommend using the RNA Clean & Concentrator-5 (R1013), which includes DNase I treatment and subsequent clean-up. This method has been validated for use with the Zymo-Seq RiboFree workflow.
Q5: How does this kit achieve probe-free, universal rRNA depletion?
The Zymo-Seq RiboFree kit uses the input RNA as a template to drive the depletion of the reverse transcribed cDNA from the highly abundant sequences. This allows the depletion to be probe-free and universally compatible with RNA from any organism. Learn more about the probe-free depletion from thisfeature in Nature.
Q6: How does this kit achieve fragmentation of the input RNA?
Generally, the random hexamer priming during reverse transcription and the reaction conditions during the depletion allows the production of appropriately sized inserts and final libraries for Illumina® sequencers.
Q7: How long should I dry the Select-a-Size MagBeads during the cleanup steps?
Humidity and temperature vary between labs. Therefore, while we don’t recommend a specific amount of time to wait, we do recommend adding the elution buffer to the bead pellet as soon as the wet gloss dries and leaves the pellet matte in appearance. By holding the tube up to a bright light, this change is more easily seen in real time.
Avoid adding the elution buffer when the bead pellet is still wet-looking, or after the pellet cracks as both may lead to a decrease in library yield. An example image showing different levels of “dryness” is here for your reference.
Q8: How can I purchase more UDI Primer Sets in addition to the included UDI 1-12 in the R3000 kit?
Besides being included in the 96-prep RiboFree kit (R3003), the UDI indexes 1-96 are also sold separately as the Zymo-Seq UDI Primer Plate (Cat. No. D3096) for you to order.
Q9: Can I prepare multiple libraries using the same UDI primer set?
Each UDI primer set can be used more than once as long as the libraries with the same UDI set ARE NOT pooled and sequenced on the same sequencing lane. For more information regarding the UDI primer sets, please refer to Appendix D in the kit protocol or contact tech@zymoresearch.com.
Q10: What sequencing platforms are compatible with this kit? Is this kit compatible with long-read sequencing?
This kit is compatible with any Illumina® sequencer. It is not compatible with the Ion Torrent®, Oxford Nanopore®, PacBio®, or other non-Illumina sequencing platforms.
Q11: Is Zymo-Seq RiboFree depletion module compatible with other RNA library prep kits?
Yes, the Zymo-Seq RiboFree depletion module is available as a stand-alone product, the Zymo-Seq RiboFree Universal cDNA Kit (R3001), and is compatible with library preparation kits that accept single-stranded DNA as an input.
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