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. 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.

Product Faq

Yes, this kit works with degraded RNA with low RIN scores, such as RNA from FFPE samples. For optimal results using degraded samples, we recommend increasing the input amount, amplifying with more PCR cycles, and adopting column cleanup for the depletion section. For details, please refer to Appendix E in the kit protocol or contact customerservice@biolinkk.com

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.

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.

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.


Front MicrobiolPublished 25 Jan 2021

‘The integrity and quality of the RNA preparations was evaluated with agarose gel electrophoresis and Agilent Bioanalyzer 2100 assays.. Total RNAseq Library Kit (Zymo Research) was used for both ribosomal RNA depletion and RNAseq library preparation, using 500 ng total RNA as input.. We constructed three libraries per RNA sample.’

VirusesPublished 12 Nov 2020

‘Total RNA quality and quantity were assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).. The Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, Irvine, CA, USA) was used for rRNA depletion and library preparation and sequencing was performed on the NovaSeq6000 (Illumina, San Diego, CA, USA) platform (150 bp paired end) at the AGRF (Melbourne, Australia).’

Genes Basel Published 20 Oct 2020

‘The resulting mixture was treated with a Turbo DNA-free kit (Thermo Fisher Scientific, Whaltham, MA, USA).. Library preparation of mRNA for high-throughput sequencing was made with R3000 Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, Irvine, CA, USA).. This kit provided the majority of CDS (coding sequences)-aligned reads.’

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“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)

“…very satisfied. It was easy and the depletion was effective.”
– Brewster K (University of Delaware)

kit components

Cat # Name Size
E2003 ZymoTaq PreMix (50 Rxns.) (2 x 625 µl)
E2004 ZymoTaq PreMix (200 Rxns.) (8 x 625 µl)
W1001-10 DNase/RNase-Free Water 10 ml
W1001-1 DNase/RNase-Free Water 1 ml
D4084-50 Select-a-Size MagBead Set 50 ml
D4084-10 Select-a-Size MagBead Set 10 ml
D3004-4-10 DNA Elution Buffer 10 ml
D3004-4-50 DNA Elution Buffer 50 ml
3DP-1002 PCR Strip MagStand 1 unit
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
R3004-1-48 Zymo-Seq Wash Buffer 48 mL
R3004-1-6 Zymo-Seq Wash Buffer 6 mL

Zymo-Seq RiboFree Total RNA Library Kit

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


Protocol: R3000
Datasheet: R3000
SDS (MSDS): R3000 | R3003