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    Zymo-Seq RiboFree Total RNA Library Kit

    R3000 / R3003

    This product was updated last year. Refer to FAQ-Q12 for details.

    Zymo-Seq RiboFree Total RNA Library Kit

    This product was updated last year. Refer to FAQ-Q12 for details.

    Cat # Name Size
    R3000 Zymo-Seq RiboFree Total RNA Library Kit 12 Preps
    R3003 Zymo-Seq RiboFree Total RNA Library Kit 96 Preps



    • Universal Depletion: Novel probe-free technology depletes rRNA from any organism.
    • Simplest Library Prep: Simultaneous ligation of both adapters reduces hands-on processing.
    • Automation Friendly: Streamlined protocol for increased scalability.

    Zymo-Seq RiboFree Total RNA Library Kit is the simplest RNA-Seq library prep kit available to make stranded, ribosomal RNA (rRNA) depleted, total RNA libraries. This all-inclusive RNA-Seq library prep kit features a novel probe-free rRNA depletion technology, RiboFree Universal Depletion, that is compatible with total RNA from any organism. Therefore, this Total RNA-Seq library kit provides a versatile and valuable tool for whole transcriptome analysis in a myriad of scientific applications. Depletion of rRNAs (which can comprise as much as 90% of a total RNA sample) is completely integrated into the library preparation workflow of this kit. RiboFree Universal Depletion uses the input RNA as templates to drive the enzymatic removal of the reverse transcribed cDNA from the overly abundant ribosomal RNA sequences, thus eliminating the need to select, design or add organism-specific probes. RiboFree Universal Depletion is validated across biological kingdom and phyla, including human, rodent, avian, plant, and various prokaryotes, as well as RNA from a wide range of sample types including cells, snap-frozen tissues, FFPE tissues, and whole blood. It’s truly One for All: Universal rRNA Depletion for Any Organism. As the simplest library preparation method for total RNA-Seq, Zymo-Seq RiboFree Total RNA Library Kit simultaneously ligates both partial P7 and P5 adapters to the first-strand cDNAs in one step without adding extra artificial sequences; also, reagents in most sections of the protocol are in premixed formats ready for use. Such convenience greatly reduces hands-on processing. The automation-friendly protocol allows RNA to NGS libraries in as little as 4 hours. Everything needed to make sequencing-ready libraries, such as unique dual indexing primers and SPRI beads, are included in the kit. It’s RNA-Seq made simple.

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    Equipment Needed (user provided) Thermal cycler with heated lid, magnetic stand for 0.2 mL PCR tubes, microcentrifuge for 0.2 mL PCR tubes and 1.5 mL microcentrifuge tubes, and a benchtop vortex mixer.

    A complimentary magnet stand is available at online checkout for direct U.S. customers of R3000 and R3003.
    Input Quality RNA should be free of DNA contamination and enzymatic inhibitors, with A260/A280 and A260/A230 ≥ 1.8. RNA with lower purity ratios (A260/A280 and A260/A230) should be treated with DNase I and purified with the RNA Clean & Concentrator™ (Cat. No. R1013) prior to processing. RNA should be suspended in water, TE, or a low-salt buffer.

    • For optimal results, please use intact RNA (RNA Integrity Number or RIN ≥ 8.0) whenever possible.
    • For degraded RNA input, see Appendix E of the protocol for additional considerations and recommended modification.
    Library Storage Libraries eluted in DNA Elution Buffer (provided) may be stored at ≤ 4°C overnight or ≤ -20°C for long-term storage.
    Processing Time As little as 4 hours
    RNA Input 10 – 250 ng of total RNA.

    • For optimal results, please use the recommended 10-250 ng input. Do not use more than 250 ng.
    • If an input below 10 ng is necessary, see Appendix D of the protocol for additional considerations and recommended modifications.
    Sample Input Material RNA from any species
    Sequencing Platform Compatibility Libraries are compatible with all Illumina® sequencing platforms except HiSeq® X.

    Illumina originally limits the applications on HiSeq X exclusively for whole-genome libraries. Please confirm with the sequencing service provider for acceptability and additional details if expecting to sequence Zymo-Seq RiboFree Total RNA libraries on HiSeq X series sequencers.
    Supplemental Info

    Purified total RNA from any organism. Please see our Multi-Organism Transcriptomics Application Note or find more product information here.

    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 100 ng as input and depleting for 2 hours yielded libraries where remaining rRNA reads were around 25%. Therefore, for rRNA depletion in metatranscriptomic samples with low complexity (<10 microbial species/strains), this kit could be suitable, and we recommend using a higher amount of total RNA as input whenever possible and optionally performing the Depletion Reaction for at least 2 hours. For further information, please contact

    It is possible to prepare libraries using this kit 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, please refer to Appendix E in the kit protocol for detailed suggestions, or contact

    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.

    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 this feature in Nature.

    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.

    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.

    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.

    Each UDI primer set in the Zymo-Seq UDI Primer Set (Cat. No. D3008) 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 B in the kit protocol or contact

    This kit is compatible with any Illumina® sequencer. It is not directly compatible with Ion Torrent®, Oxford Nanopore®, or PacBio® platforms. 

    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.

    There are many ways for you to be certain about which instruction manual to follow. One way is to check the cold reagent names: they are different between the two versions, and the updated version includes a reagent called "Depletion Reagent 4". Overall, compared to v1.3.0, the updated version has a lower minimum input (10 ng total RNA), a shorter depletion reaction time (1 hour for 100 ng of input), and one fewer round of bead cleanup. To learn more of the kit features since the update, or if you need an electronic copy of the previous instruction manual (v1.3.0), please contact

    It is possible to prepare libraries with input lower than 10 ng, though the performance may be negatively impacted. For modifications of the protocol when using 1-9 ng of RNA as input, please refer to Appendix D in the kit protocol for detailed suggestions or contact We do NOT recommend extending the depletion reaction to run overnight.

    Library yield can vary depending on factors such as the quality and quantity of the input RNA, and the cycle number of the PCR reaction for library amplification. In general, using 10 ng of Universal Human Reference RNA (RIN > 8.0) as input, the library yield is > 10 nmol/L when amplified with 12 PCR cycles and eluted to 20 µL of DNA Elution Buffer.

    “This is probably the best RNA-seq library preparation kit I’ve used (and I have used many). It is very simple and gives clean, reproducible results at a very affordable price. I would highly recommend this product to anyone looking for a quicker and easier library preparation kit. Zymo has never let me down.”

    - Galina (City of Hope)

    “I think this kit is faster and simpler to do than the Truseq RNA library prep kit. It was easy enough that I tried it once and got a nice library prep, with a high concentration and I did not make any mistakes in my first try! I will definitely buy this kit and recommend it to others without a doubt.”

    - Rafael (UC Berkeley)

    “This RNA Library Kit was easy to use and recovered good efficiency.”

    - Veronica L (CICY Mexico)

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    Cat # Name Size
    R3001 Zymo-Seq RiboFree Universal cDNA Kit 12 Preps
    D3008 Zymo-Seq UDI Primer Set (Indexes 1-12) 12 indexes
    D3096 Zymo-Seq UDI Primer Plate (Indexes 1-96) 96 Indexes
    R3004-1-6 Zymo-Seq Wash Buffer 6 mL
    R3004-1-48 Zymo-Seq Wash Buffer 48 mL
    D3004-4-10 DNA Elution Buffer 10 ml
    D3004-4-50 DNA Elution Buffer 50 ml
    W1001-1 DNase/RNase-Free Water 1 ml
    W1001-4 DNase/RNase-Free Water 4 ml
    W1001-6 DNase/RNase-Free Water 6 ml
    W1001-10 DNase/RNase-Free Water 10 ml
    W1001-30 DNase/RNase-Free Water 30 ml
    3DP-1002 PCR Strip MagStand 1 unit

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