NEXTFLEX® Small RNA-Seq Kit v4 for Illumina® Platforms

  • Completely gel-free protocol from purified miRNA or 1ng of total RNA
  • Completely automated protocol on the PerkinElmer® Sciclone® G3 NGSx, Sciclone® G3 NGSx iQ™, and the Zephyr® G3 NGS workstation
  • Patent pending technology optimized for challenging samples such as plasma or serum, including exosomes
  • Exceptional miRNA discovery
  • Ready to sequence libraries in ~ 6 hours
  • Multiplexing of up to 384 samples
  • An automation protocol is available
  • Functionally validated with Illumina® sequencing platforms
Download Manual
Looking for NEXTFLEX Small RNA-Seq Kit v3, click here
For research use only. Not for use in diagnostic procedures.

  • NOVA-5132-31

    8 RXNS | UDI 1-8

  • NOVA-5132-32

    48 RXNS | UDI 1-48

  • NOVA-5132-41

    96 RXNS | UDI 1-96

  • NOVA-5132-42

    96 RXNS | UDI 97-192

  • NOVA-5132-43

    96 RXNS | UDI 193-288

  • NOVA-5132-44

    96 RXNS | UDI 289-384

Gel-Free Small RNA Library Prep with Reduced Bias

The NEXTFLEX® Small RNA-Seq Kit v4 uses patent-pending technology to provide a completely gel-free small RNA library preparation solution for Illumina® sequencing platforms. Our approach to reducing bias involves optimization of the ligation reaction and the use of dimer reduction oligonucleotides. These changes results in decreased bias in comparison to NEXTFLEX® Small RNA-Seq Kit v3 and standard protocols.

The legacy NEXTFLEX Small RNA-seq kits v3 are still available and can be found here.

NEXTFLEX® Small RNA-Seq Kit v4 allows preparation of libraries ready for sequencing with a simplified workflow in approximately 5 hours, with as little input as 1 ng of total RNA. Libraries prepared with this kit have a higher proportion of mapping reads and unique miRNAs than leading competitors (Figure 1 and 2).

Gel-Free small RNA library prep with increased discovery rates
Figure 1
Gel-free small RNA library prep from serum
Figure 2

Maximize miRNA Mapped Reads with NEXTFLEX® Blood miRNA Blockers

The NEXTFLEX® Blood miRNA blockers deplete common miRNA in blood and plasma such as miR-486-5p, miR-92a-3p and miR-451a. These constitute 50-70% of the small RNA present in those samples and usually are not of interest for researchers.

Small RNA-Seq Library Prep Optimized for Automation

By eliminating all gel-based steps even for low input samples, the NEXTFLEX® Small RNA-Seq Kit V4 is optimized for easy migration onto automated liquid handling platforms. Currently methods are available for the PerkinElmer Sciclone® G3 NGSx, Sciclone G3 NGSx iQ™, and the Zephyr® G3 NGS workstation.

For more information contact [email protected].

Illumina® Small RNA-Seq Multiplexing with UDI Barcodes

Up to 384 UDI barcoded primers are included in each kit for multiplexing. These UDIs were redesigned, allowing libraries prepared with this kit to be run on any Illumina® sequencing platform. These include the Illumina® NextSeq® and Novaseq® sequencers and only require standard Illumina® sequencing primers.


  • NEXTFLEX® 3’ Adenylated Adapter v4
  • NEXTFLEX® 3’ Ligation Buffer v4
  • NEXTFLEX® 3’ Ligation Enzyme Mix v4
  • NEXTFLEX® Adapter Depletion Solution
  • NEXTFLEX® tRNA/YRNA Blockers v4
  • NEXTFLEX® Adapter Inactivation Mix v4
  • NEXTFLEX® 5’ Adapter v4
  • NEXTFLEX® 5’ Ligation Buffer v4
  • NEXTFLEX® 5’ Ligation Enzyme Mix v4
  • NEXTFLEX® RT Enzyme Mix v4
  • NEXTFLEX® RT Buffer v4
  • NEXTFLEX® RT Primer v4
  • NEXTFLEX® UDI Barcoded Primer Mix v4 (6.25 μM, 4 μL per well)
  • NEXTFLEX® Small RNA PCR Master Mix v4
  • Resuspension Buffer
  • Nuclease-free Water
  • microRNA Control
  • NEXTFLEX® Cleanup Beads


  • 1 ng – 2 μg total RNA or purified small RNA from 1-10 μg total RNA in up to 5 μL Nuclease-free Water
  • Isopropanol
  • 80% Ethanol
  • 2, 10, 20, 200 and 1000 μL pipettes
  • RNase-free pipette tips
  • Microcentrifuge
  • 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
  • Thin-wall nuclease-free PCR tubes
  • Thermal Cycler
  • Vortex
  • Magnetic Stand -96 (Ambion, Cat # AM10027) or similar


  • Sequences of NEXTFLEX Small RNA-Seq Barcode Indexes – Excel


The NEXTFLEX® Small RNA Sequencing Kit v4 contains the reagents, including barcoded primers, to prepare 8, 48, or 96 small RNA samples for Illumina® next-generation sequencing. The shelf life of all reagents is 6 months when stored properly. All components can safely be stored at -20°C, except: NEXTFLEX® Adapter Depletion Solution, Resuspension Buffer, Elution Buffer, and Nuclease-free Water, which can be stored at room temperature, and NEXTFLEX® Cleanup Beads, which should be stored at 4°C.

Components ship on dry ice and at room temperature.

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