Reduced dimer formation means no adapter titration

Adaptase technology results in minimal adapter dimers, which means no need for adapter titration (Figure 2). Compared to leading RNA library kits, which can produce libraries with >10% adapter dimers and require adapter titration steps, the xGen Broad-Range RNA Library Prep Kit produces <1% adapter dimers and maintains ligation efficiency at all supported input levels.

Figure 2. Comparison of kits. Libraries were prepared using two different kits with the same quantity of input material (n = 1 per input quantity) and subjected to the same number of PCR cycles according to the xGen Broad-Range protocol recommendations during library amplification. Representative library sizes, yields, and BioanalyzerTM (Agilent) traces illustrate typical libraries generated from the same input series of poly(A)-enriched Universal Human Reference (UHR) RNA (Agilent 740000) or human brain mRNA (Takara 636102), when processed by either the xGen Broad-Range RNA Library Prep Kit (left panel) or an RNA library kit from an alternate supplier (right panel). The arrows indicate adapter dimers that were generated during library preparation.

High mapping and transcript identification with lower duplication rates

Figure 3. Comparison of data obtained using different suppliers’ kits. Universal Human Reference (UHR) Total RNA (Agilent 740000) was enriched using the NEBNext® poly(A) mRNA Magnetic Isolation Module (NEB E7490), before being processed by the xGen Broad-Range RNA Library Prep Kit and other supplier kits K and N. For each kit evaluated, libraries were prepared at 10, 100 and 500 ng input, where n = 1 sample per input quantity. PCR amplification of each library was performed as follows: 10 cycles for 500 ng inputs, 12 cycles for 100 ng inputs, and 16 cycles for 10 ng inputs. Libraries were sequenced on a MiniSeqTM with 2x75 bp paired-end reads. Fastq files were downsampled to 2.2 million reads before analysis using STAR (mapping rate), RNASeqC (genes/transcripts detected), or Picard (duplication rate). The xGen Broad-Range RNA Library Prep Kit has high mapping percentage, detects more genes and transcripts, and has fewer duplicates.

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).

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).

The xGen Broad-Range RNA Library Prep Kit has a fast NGS transcriptomics research workflow with comprehensive transcript coverage and NGS data quality from a broad range of input quantities for Illumina® sequencing platforms. Leveraging proprietary AdaptaseTM technology, this 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. The kit is compatible with manual and automated workflows, as well as upstream and downstream enrichment and depletion methods. It supports a variety of indexing options in research studies.

Figure 1. xGen Broad-Range Library Prep workflow: After RNA fragmentation, the reverse transcriptase step uses random primers to generate the first-strand cDNA. Next, Adaptase technology simultaneously performs tailing and ligation to incorporate the R2 Stubby Adapter to the 3’ ends of the cDNA molecules. The extension step produces a dsDNA duplex, while ligation adds the R1 Stubby Adapter to the 3’ ends of the primer-extended cDNA molecules. Finally, indexing PCR increases library yield, incorporates single or dual indexes, and results in full-length adapters at the ends of each molecule. In addition, bead-cleanup steps are needed after extension, ligation, and final indexing PCR steps. 

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.

• Comprehensive data—for mapping rates, genes identified, and transcript coverage.
• Accommodates your samples with a single kit—consistent results from 10 ng to 1 µg total RNA or 100 pg to 100 ng mRNA input.
• Consistent libraries—minimal adapter dimers so adapter titration is not required for supported inputs.
• Save time, reduce costs—go from RNA to library in 4.5 hours with a wide variety of index options, ranging from up to 1536 UDI primer pairs (available with or without NormalaseTM), and up to 96 CDI primer pairs (with or without Normalase).
• Workflow—designed for easy automation.

xGen™ Broad-range RNA Library Prep Kit

RNA-Seq library prep kit that supports total RNA inputs from 10 ng to 1 µg, includes cDNA synthesis through library prep and amplification; (upstream modules: poly(A)-selection beads, ribodepletion and indexing primers sold separately).

xGen™ Normalase™ Module

Core kit for performing Normalase, includes Normalase I and Normalase II reagents and Normalase terminal primers, Normalase indexing primers sold separately.

xGen™ Normalase™ UDI Primers

Premixed, unique dual index primers designed for Normalase™ workflow. Individual plates of 96 indexes or sets of 384 indexes are available. Or choose all four sets for a total of 1536 index pairs. All plates are single-use to minimize the risk of contamination by multiple handling events.

xGen™ UDI Primer Pairs

Premixed unique dual index primer pairs for sample indexing by PCR. These are delivered as single-use plates containing either 16 or 96 primer pairs, or for high-volume/high-throughput customers we have bulk 2 nmol offerings of primer with 10 nt indexes. The 10 nt indexes are available in sets of 384, 768, and 1536 index pairs. All plates except the bulk 2 nmol offerings are single-use to minimize the risk of contamination by multiple handling events.

Legacy Swift Indexing Primers

Legacy Swift Primers, CDI and UDI indexing strategies. The xGen™ CDI Primers are individual tubes of indexes, to be arrayed in a combinatorial fashion for up to 96 possible barcode combinations, and the xGen™ UDI Primers, Plate 2 are premixed Unique Dual Index primers in single-use plates. Both products have 8nt barcodes. Note that these primers are not compatible with any other IDT indexing primers, due to potential demultiplexing issues.

xGen™ Normalase™ CDI Primers

Combinatorial dual index primers designed for Normalase workflow. Consists of 20 individual tubes of indexes that support up to 96 combinations of i7-i5 indexes.

xGen™ RNA Library Prep Kit

RNA-Seq library prep kit that supports total RNA inputs from 100 ng to 1 µg, includes cDNA synthesis through library prep and amplification; (upstream modules: poly(A)-selection beads, ribodepletion, and indexing primers sold separately) faster workflow with fewer steps than the xGen Broad-Range RNA Library Prep Kit.

 xGen Broad-Range RNA Library Prep Kit Protokoll

IDT NGS termékkatalógus

xGen Normalase Module Protokoll

xGen RNA Library Prep Kit Protokoll