The SEQuoia Complete Stranded RNA Library Prep Kit is a high-performance RNA-Seq kit that captures long and short RNAs in a single library, even from limited and low-quality samples. The streamlined workflow minimizes the number of pipetting steps and reduces the overall protocol time to less than 4 hours. The kit allows construction of libraries that are >99% stranded and suitable for next-generation sequencing on Illumina platforms and includes access to SEQSense Analysis Solution and Toolkit, a dedicated secondary data analysis pipeline.
Featuring SEQzyme, a proprietary engineered enzyme that couples cDNA synthesis with adapter addition in a continuous synthesis reaction. The unique enzymatic properties effectively captures all types and sizes of RNA species in a novel enzymatic reaction, significantly improving the diversity and quality of RNA libraries, even from limited or degraded RNA samples.
Features and Benefits
Experience the Power of SEQzyme
Highly Efficient and Streamlined Library Prep Workflow
The unique enzymatic properties of SEQzyme produce robust, high-quality RNA library. The kits are optimized to facilitate efficient library synthesis using a wide range of input RNA (100 pg to 1 μg) and are compatible with automated next-generation sequencing library construction. The streamlined workflow minimizes the number of pipetting steps and greatly reduces the overall protocol time to less than 4 hours.
Diverse Libraries with Low Quality and Limited Input Samples
The kit constructs a stranded (>99%) library that captures all RNA biotypes, both long and short RNAs, even from poor quality RNAs (for example, FFPE tissue samples). The feature enables researchers to discover biologically relevant differences in more areas of the transcriptome.
Dedicated Data Analysis Toolkit
SEQSense Analysis Solutions and Toolkit, which is available through a web portal, or can be downloaded and run locally, enables users to trim raw sequencing data and align sequences to a reference genome. The output provides specific quality control metrics to assess the quality of the sequencing data as well as a BAM file, which can be used with tertiary analysis packages to perform advanced analyses.
Applications and Uses
- Differential gene expression and whole transcriptome profiling of high- and low-quality RNA samples
- Sorted or single cell
- Characterization of non-polyadenylated RNAs
- Gene fusion identification
- Single nucleotide variation discovery
- Biomarker discovery
- Splice junctions identification
Each kit comes with 2 reagent boxes. Box A includes reagents for RNA fragmentation, cDNA synthesis, and library preparation. The kit is stored at –20°C:
- Reagent A Fragmentation Mix
- Reagent B End Repair Enzyme
- Reagent C Poly(A) Buffer
- Reagent D Poly(A) Polymerase
- Reagent E SEQzyme Mix
- Reagent F Amplification Mix
Box B includes magnetic bead for purification. The kit is stored at 4°C
SEQuoia Dual Indexed Primers are available and can be ordered separately.
SEQuoia Complete RNA Library Preparation Kit FAQs
Kit Contents and Storage
The SEQuoia Complete RNA Library Prep Kit provides all reagents required to construct a library from RNA, with the exception of the SPRISelect beads from Beckman Coulter.
|Component||Cap Color||24 Reactions||96 Reactions||Storage Temperature|
|Reagent A Fragmentation Buffer||Red||48 μl||192 μl||- 20 °C|
|Reagent B End Repair Enzyme||Yellow||48 μl||192 μl||- 20 °C|
|Reagent C Poly(A) Buffer||Violet||570 μl||2x 1.1 ml||- 20 °C|
|Reagent D Poly(A) Polymerase||Blue||30 μl||120 μl||- 20 °C|
|Reagent E SEQzyme Mix||Orange||120 μl||480 μl||- 20 °C|
|Reagent F Amplification Mix||Green||600 μl||2x 1.2 ml||- 20 °C|
|Purification Beads||Grey||480 μl||2x 960 μl||4 °C|
The following reagents are required but not supplied. These materials have been validated with the SEQuoia Complete Stranded RNA Library Prep Kit protocol. Substitutions may not produce ideal results.
- 2 ml RNase, DNase free, low-bind PCR tube strips and cap strips (TBC0802 or TBC1202)
- RNase-free filtered pipet tips: 10 μl, 20 μl, and 200 μl
- SPRISelect beads from Beckman Coulter
- 10mM Tris-HCl pH8 (RNase, DNase Free)
- 80% ethanol (prepare fresh)
Optional Materials Not Provided
- SEQuoia Dual Indexed Primers Set (12011928) or SEQuoia Dual Indexed Primers Plate (12011930)
- rRNA depletion kit
- ddPCR Library Quantification Kit for Illumina TruSeq (1863040)
- Thermal cycler for accurate incubation temperatures
- Calibrated single-channel and multi-channel pipets: 10 μl, 20 μl, and 200 μl
- Magnetic rack for small-scale separation of magnetic beads in 0.2 ml tubes
- Microcentrifuge for 0.2 ml tubes
- 96-well PCR cooler rack
- Ice bucket
The ideal RNA purification strategy depends on the sample type and experimental objectives. We have tested and recommend using PureZOL RNA Isolation Reagent (7326880) for tissue samples and the SingleShot Cell Lysis Kit (1725080) for cultured cells. If small RNAs are of interest, we do not recommend using a column-based based kit, as RNA fragments under 200 may be lost. Instead, use the SingleShot Cell Lysis Kit.
Additionally, these references provide a good overview of various methods for small RNA isolation;
- Quantiﬁcation of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Proﬁles. Masotti A, et al. Journal of Biomedicine and Biotechnology Volume 2009, Article ID 659028
- A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. Sellin Jeffries MK, Kiss AJ, Smith AW, Oris JT. BMC Biotechnology, 201414:94
The SEQuoia Complete Stranded RNA Library Prep kit is compatible with 1 ng to 1 μg of total RNA (pre-depleted of ribosomal RNA)
RNA Library Amplification
Typically, SEQuoia Complete Stranded RNA Library Prep Kit requires 3–5 fewer cycles compared to other commercially available kits, due to a high conversion rate. This is advantageous because over amplification can result in artifacts such as amplification bias, increased duplicate rates, and chimeras. To minimize artifacts, optimize the number of PCR amplification cycles so that the final library concentration is between 200–1,000 ng. The number of PCR cycles is dependent upon the amount and quality of input RNA. The following table provides general guidelines and can be used as a starting point for optimization.
PCR Cycle Recommendations
|Input Amount Total RNA||# of PCR cycles|
SEQuoia Complete Stranded RNA Library Prep Kit captures both short and long RNA biotypes, including mRNA, miRNA, lncRNA, snoRNA. Because this kit efficiently captures small RNAs, it is recommended that sequencing depth be 20M–30M reads to ensure the accuracy of the results.
Sequencing and Data Analysis
We recommend sequencing with single-end reads to save on sequencing cost.
- The first 8 bases on R2 are a random tag sequence which can be used as a UMI.
- Recommended read length: 100 x 8 or 75 x 8 of Read1 x Read2
- Read depth should be determined based on the application and prior experience. The typical range is 20–30M single-end reads due to the number of small RNA fragments captured, which increases the library complexity.
- With single reads, using SEQuoia Complete Stranded RNA Library Prep Kit, you can get coverage of full transcript as shown with all the data on this page.
- For pair-end reads, there may be a significant drop in read quality for R2 because of the presence of the poly(T) sequence.
No. The adapters in the SEQuoia Complete Stranded RNA Library Prep Kits are compatible with flow cells used with Illumina platforms.
|17005726||SEQuoia Complete Stranded RNA Library Prep Kit||24 reactions|
|17005710||SEQuoia Complete Stranded RNA Library Prep Kit||96 reactions|
|12011928||SEQuoia Dual Indexed Primers Set, 12 vials of Unique Dual Indexes||96 reactions|
|12011930||SEQuoia Dual Indexed Primers Plate, one 96-well plate of Unique Dual Indexes||96 reactions|
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A high-performance RNA sequencing (RNA-Seq) kit that captures long and short RNAs in a single library, even from limited and low-quality samples. The kit provides all of the components required to make an RNA-Seq library for Illumina platforms. The size of the kit is for 24 reactions.
A high-performance RNA sequencing (RNA-Seq) kit that captures long and short RNAs in a single library, even from limited and low-quality samples. The kit provides all of the components required to make an RNA-Seq library for Illumina platforms. The size of the kit is for 96 reactions
For use with SEQuoia Complete Stranded RNA Library Prep Kits. Contains 12 unique dual indexed primers supplied in 12 separate vials, for 96 reactions
Pkg of 20, bag of 12 x 8 thin-wall polypropylene tube strips and 12 x 8-cap strips for PCR, natural (1,920 tubes and 1,920 caps)
Pkg of 20, bag of 8 x 12 thin-wall polypropylene tube strips and 8 x 12-cap strips for PCR, natural (1,920 tubes and 1,920 caps)