Bio-Rad, 17005726, SEQuoia Complete Stranded RNA Library Prep Kit, 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 24 reactions.
Overview
Frequently Asked Questions
Learn more about how this product is being used.
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.
Experience the Power of SEQzyme
Features and Benefits
• Captures long and short RNAs in a single prep
• Capable with low sample input requirements
• Validated with FFPE and other low-quality samples
• Full protocol can be completed in less than 4 hours
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.
Applications and Uses
• Differential gene expression and whole transcriptome profiling of high- and low-quality RNA samples Sorted or single cell Micro-dissection Characterization of non-polyadenylated RNAs Gene fusion identification Single nucleotide variation discovery
• Sorted or single cell
• Micro-dissection
• Characterization of non-polyadenylated RNAs
• Gene fusion identification
• Single nucleotide variation discovery
• Biomarker discovery
• Splice junctions identification
Included Components
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
What is included in a SEQuoia Complete Stranded RNA Library Prep Kit?
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.
Are there required materials that are not included in the kit?
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 )
Equipment List
• 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
• Vortex
• Microcentrifuge for 0.2 ml tubes
• 96-well PCR cooler rack
• Ice bucket
Sample Preparation
Which RNA isolation method or kit should I use?
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
What range of input total RNA can be used in a library prep?
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
How many PCR cycles are recommended for 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
What molecules are captured by SEQuoia Complete Stranded RNA Library Prep Kit?
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
What sequencing parameters are recommended?
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.
Is SEQuoia Complete Stranded RNA Library Prep Kit compatible with the Ion Torrent platform?
No. The adapters in the SEQuoia Complete Stranded RNA Library Prep Kits are compatible with flow cells used with Illumina platforms.
See All SEQuoia FAQs »
Configuration Options
Related Products
• PureZOL RNA Isolation Reagent
• SingleShot Cell Lysis Kit
• C1000 Touch Thermal Cycler
• ddPCR Library Quantification Kit for Illumina TruSeq