Processing
xGen™ RNA Library Preparation Kit
Accelerate your RNA-Seq transcriptomics research
The xGen RNA Library Prep Kit offers a fast NGS transcriptomics research workflow. Assemble libraries by either manual or automated workflow directly from 1st strand cDNA synthesis while using your preferred indexing options for the experiment.
xGen NGS—made for RNA library preparation.
Ordering
- Save time and cost—results in as little as 3.5 hours. Combined with the lower cost, you can process more samples in less time to increase your data output.
- Supports many workflows with indexing options, automation, and Normalase™ Technology—combinatorial dual indexing up to 96-plex, unique dual indexing up to 1536-plex, and enzymatic library normalization available; automation-friendly.
- Reliable functionality—maintains ligation efficiency at all supported inputs and no adapter titration required.
Product details
The xGen RNA Library Preparation Kit offers a fast, low-cost transcriptome sequencing workflow for Illumina® platforms. Our RNA library prep kit enables stranded RNA library construction directly from 1st strand cDNA without the requirement for 2nd strand cDNA synthesis and degradation, or template-switching methods. This easily automatable kit is compatible with upstream and downstream enrichment and depletion methods and supports a variety of indexing options. These include upstream mRNA enrichment and rRNA depletion modules as well as downstream xGen Hybridization capture workflows for targeted RNA sequencing.
Table 1. Research applications
| Application | xGen RNA Library Prep Kit |
|---|---|
| Gene expression profiling | ✓ |
| Research on disease-associated transcripts | ✓ |
| Total RNA + hybridization capture enrichment | ✓ |
Automation
The xGen RNA Library Prep Kit protocol is readily automatable. A 10% overage volume of reagents is supplied to accommodate automation, and additional reagent overage volume is available upon request.
Table 2. Product specifications
| Feature | Specification | Benefit |
|---|---|---|
| Input quantity | 100 ng to 1 μg total RNA 5 ng to 100 ng mRNA | Supports a wide input range |
| RNA types supported | Poly(A)-enriched mRNA Ribo-depleted RNA Total RNA | Supports most RNA applications |
| Technology | Adaptase™ tailing and ligation to 1st strand cDNA | Maintains strandedness (≥97%) No 2nd strand cDNA No adapter titration |
| Workflow time* | 3.5 hours | Less hands-on time |
| Kit reaction sizes | 16, 96, and 4x96 | Evaluation and adoption |
| Components provided | Fragmentation module RT module Library prep Polymerase | Complete solution for processing total, enriched, or depleted RNA from transcript to library |
| Indexing options | Combinatorial dual Unique dual Normalase | Flexible to sequencers, workflows, and applications |
| Multiplexing capability | Up to 1536 libraries | Save sequencing costs |
| Automation | Compatible with liquid handlers Custom packaging available | Adopt at scale |
*Workflow time is based upon incubation times and expected times for hands-on-steps. Actual workflow time may vary depending on individual factors in your laboratory.
Workflow
Reverse transcription—During this step, RNA fragmentation, reverse transcription, and incorporation of the R1 Stubby Adapter are performed, followed by a purification step.
Adaptase technology—This step performs 3’ tailing and ligation of the R2 Stubby Adapter to first strand cDNA
Indexing PCR—This step completes adapter sequences, adds sample-specific index sequences, and increases library yield, followed by a purification step.
Figure 1. xGen RNA Library Prep Kit workflow converts RNA directly into an indexed library. The workflow for creating an RNA-seq library using the xGen RNA Library Prep Kit includes a proprietary Adaptase technology step that adds a single-stranded R2 Stubby Adapter onto the 3’ end of the 1st strand cDNA product. Indexing primers are then used to amplify the library and add the full-length adapter sequence. See text for details.
Index and kit options
The xGen RNA Library Prep Kit uses primers that anneal to the Stubby Adapters added during the random priming and Adaptase steps, to be fully compatible with Illumina® sequencers. IDT supplies a variety of index configurations and strategies, including:
- Combinatorial dual indexing, up to 96 combinations
- Unique dual indexing, up to 1536 unique dual indices
Product data
Save time with direct conversion of 1st strand cDNA
Incubation times for enzymatic and purification steps are shown according to vendor supplied user instructions.
Figure 2. Comparison of different workflow times for RNA-seq library preparation kits. The xGen RNA Library Prep Kit constructs library molecules directly from 1st strand cDNA synthesis, which reduces the time from fragmentation (F) to clean-up (C).
Reliable mapping and strandedness
The xGen RNA Library Kit shows high mapping rates and maintenance of strandedness.
Figure 3. Reliable mapping rate and strandedness with the xGen RNA Library Prep Kit. (A) Universal Human Reference (UHR) Total RNA (Agilent) was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Libraries were constructed from 50, 100, and 250 ng of enriched mRNA using the xGen RNA Library Prep Kit and the Supplier K’s kit according to the manufacturer’s instructions. (B) Human Brain mRNA (Clontech) was used to construct the xGen RNA and the Supplier K’s libraries from 1, 5, 10, 50, 100 ng of Human mRNA. The percent strandedness was determined by the average of the five input quantities. For both graph A and graph B, libraries were sequenced on a MiniSeqTM with 2 X 75 bp paired-end reads. Fastq files were downsampled to 3.8 million reads before analysis using STAR (mapping rate) or RNA-SeQC (strandedness).
Resources
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.
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