Highlights
- 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.
Description
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.
Table 1. Zymo-Seq RiboFree Total RNA Library Kit (Cat. No. R3000/R3003) update summary
| | Current Version | Updated Version |
Instruction Manual Versions | v1.3.0 | v2.0.0 |
Minimum Total RNA Input | 100 ng | 10 ng |
Depletion Reaction Duration* | 100 ng input – 4 hours | · 100 ng input – 1 hour
· 10 ng input – 4 hours |
Hands-on Time | 4 rounds of bead cleanup | 3 rounds of bead cleanup |
* May vary depending on the quantity, quality, and source of the input RNA.
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 | - Any Organism. One rRNA Depletion Solution.
- Evaluating Quality of Input RNA for NGS Library Preparation
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