NEXTFLEX Small RNA Sequencing Kit v4 (96rxn, BC1-96)

Product Description

Generate high-quality, gel-free small RNA libraries with reduced bias and minimal input, enabling robust biomarker discovery and assay development on Illumina® sequencing platforms. This NEXTFLEX Small RNA Sequencing Kit v4 contains 96 UDI Barcodes.

Key Benefits

• Automation ready workflow and 384 available UDI barcodes
• Completely gel free protocol from 1 ng total RNA
• Bias and dimer reduction via optimized ligation and dimer reduction oligos
• High miRNA discovery and mapping performance vs. competitors
• Optimized for challenging biofluids and exosomal RNA
  
  

Why you should use NEXTFLEX® Small RNA-Seq

Bias-reduced small RNA libraries for confident profiling

The NEXTFLEX® Small RNA-Seq Kit v4 with UDIs is designed to prepare small RNA libraries from total RNA or purified small RNA while greatly reducing sequence bias and adapter-dimer formation. By optimizing the ligation reaction and incorporating dimer reduction oligonucleotides, the kit delivers decreased bias compared to standard protocols, enabling more accurate identification and quantification of microRNAs, piRNAs and other small RNAs.

Libraries generated with this kit have a higher proportion of mapping reads and unique miRNAs than leading competitors, supporting reliable detection of low-abundance species that are critical for biomarker discovery and translational assay development.

-Dr. Sören Franzenburg, Head of NGS platform, Kiel University

Gel-free, automation-ready workflow

This kit provides a fully gel-free small RNA library preparation workflow for Illumina® sequencing, even from low-input samples such as 1 ng of total RNA. The streamlined protocol includes 3’ adenylated adapter ligation, excess adapter depletion, 5’ adapter ligation, reverse transcription, PCR amplification and size selection using bead-based cleanup steps, eliminating labor-intensive gel extractions. The complete protocol can be finished in approximately 5–7 hours, with defined safe stopping points to fit into busy lab schedules.

By removing all gel-based steps, the workflow is optimized for seamless migration to automated liquid handling platforms, with validated methods available for the Revvity Sciclone® G3 NGSx, Sciclone® G3 NGSx iQ™ and Zephyr™ G3 NGS workstations.

Flexible input range and optimized for challenging sample types

The NEXTFLEX® Small RNA-Seq Kit v4 with UDIs has been validated with 1 ng–2 μg total RNA, RNA isolated from plasma and serum, and synthetic miRNA pools, providing flexibility across discovery and preclinical sample types. It supports cell-free RNA, including RNA isolated from plasma, and is optimized for difficult matrices such as plasma, serum and exosomes.

Included NEXTFLEX® tRNA/YRNA Blockers v4 deplete abundant tRNA and YRNA fragments commonly found in cell-free RNA, increasing sequencing depth for informative small RNAs. For project-specific needs, NEXTFLEX® Custom Small RNA Blockers enable selective depletion of up to 10 high-abundance sequences of the user’s choice, maximizing discovery of biologically relevant transcripts.

High-throughput multiplexing with UDI protection

Sequencing libraries generated with this kit incorporate Unique Dual Indices (UDIs) specifically designed to address index hopping on Illumina® platforms with patterned flow cells. These UDIs help prevent mis-assigned reads in multiplexed runs, providing high assurance of data integrity in high-throughput studies. NEXTFLEX small RNA sequencing kits include up to 384 UDI barcoded primers for extensive multiplexing capacity.

The redesigned index architecture ensures that libraries prepared with this kit are compatible with all Illumina® sequencing instruments, including MiSeq®, HiSeq® 2000/2500, MiniSeq®, NextSeq® and NovaSeq® systems, using standard Illumina® sequencing primers. The libraries are also compatible with Element® sequencing platforms, broadening instrument flexibility for core and production environments.

Multiplex up to 1,536 samples on a single flow cell with NEXTFLEX™ Unique Dual Index Barcodes (10NT UDIs). Off-the-shelf plate sets (containing between 16 to 1,536 unique adapters) let you start small and scale to population-level projects without requiring custom ordering or re-balancing.
Browse available NEXTFLEX NGS UDI Barcodes.

How it Works

Figure 1: Sample flow chart

Supporting Data

Figure 2: NEXTFLEX Small RNA-Seq Kit v4 increases miRNA reads while reducing tRNA/YRNA carry-over. Libraries from 1 ng plasma RNA were sequenced and aligned to miRNA, tRNA, and YRNA reference sets. Compared with a competitor workflow, the NEXTFLEX Small RNA kit aligns with more miRNAs while, while minimizing reads associated with t/YRNA and RNA, improving effective sequencing depth for downstream small-RNA analysis.

Figure 3: NEXTFLEX Small RNA-Seq Kit v4 detects higher number of unique miRNA species across different sample types.

Figure 4: NEXTFLEX Small RNA v4 identifies 45% more unique miRNA species than competitor workflows. Venn diagram of libraries prepared from 1 ng serum RNA shows 81 miRNAs detected with both kits, an additional 53 miRNAs unique to NEXTFLEX, and 11 unique to the comparator.

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