Zymo-Seq RiboFree Total RNA Library Kit

Zymo-Seq RiboFree


  • The Fastest & Easiest Total RNA-Seq Library Kit: Prepare stranded, RiboFree libraries from total RNA in 3.5 hours.
  • Compatible with Any Organism: Probe-free technology depletes rRNA & Globin from any RNA source.
  • The Most Accurate: Eliminate bias from rRNA depletion.


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 and globin 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, globin, or any other over-abundant, repetitive transcript 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 and globin, resulting in minimal off-target effects as low as 1.8% in all protein-coding transcripts.

This total RNA-Seq library prep kit’s protocol is fragmentation-free and works with up to 2 ug of undepleted total RNA, down to 50 ng of undepleted total RNA, or as little as 5 ng of ribosome-depleted RNA samples. 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, SPRI beads, and magnetic bead stand are included in the kit. It’s RNA-Seq made simple.

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Technical Specifications

Equipment Required Thermocycler, magnet stand*, and microcentrifuge.

*for a complimentary magnet stand, please contact tech@zymoresearch.com

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.

Product FAQ

Q1: Do you sell the ribo depletion separately?

Yes, please contact technical support tech@zymoresearch.com for ordering information.

Q2: Will this kit work with prokaryotes?

Yes, this kit will work with any organism. As long as your sample has rRNA, this kit will work.

Q3: My sample is lower than 100 ng, will this kit still work?

Lower sample inputs will require a longer incubation time during RiboFree Universal Depletion. Please refer to Appendix B for suggestions



“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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Kit Components

Zymo-Seq RiboFree Total RNA Library Kit

R3000 / R3003

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


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