Multiplexing up to 96 RNA Libraries for Illumina® Sequencing

The patent pending  NEXTFLEX® Rapid RNA-Seq Kits for Illumina® RNA-seq library prep provide an easy and flexible solution for generating single end or paired-end libraries as well as a variety of multiplexing options. This kit incorporates the NEXTFLEX® Rapid Reverse Transcriptase, a robust, thermostable RT that executes cDNA conversion with robust yields.

This kit was designed to be used in conjunction with the NEXTFLEX® RNA-Seq Barcodes or NEXTFLEX-96™ RNA-Seq Barcodes for multiplexing. The availability of up to 96 unique adapter barcodes makes this the most high-throughput kit available for RNA library prep.

Automated RNA Library Prep Protocols

An optimized automation protocol for RNA library prep using the NEXTFLEX® Rapid RNA-Seq Library Prep Kit are available for the Sciclone® NGSx workstations is now available.

Magnetic Beads for mRNA Purification

The NEXTFLEX® Poly(A) Beads 2.0 now provide a convenient method for batch purification of pure, intact mRNA upstream NEXTFLEX® Rapid RNA-Seq library preparation. NEXTFLEX® Poly(A) Beads 2.0 use oligo(dT) primer to isolate polyadenylated messenger RNAs from 10 ng – 5 µg of previously isolated total RNA.

Features

• Faster than traditional Illumina® RNA library prep protocols

• Complete solution includes thermostable NEXTFLEX® Rapid Reverse Transcriptase

• Input – 10 ng – 1 µg total RNA for enrichment by NEXTFLEX® Poly(A) Beads 2.0 or ~ 1 ng – 100 ng isolated mRNA or rRNA-depleted RNA

• 96 barcodes are available for multiplexing

• Automation protocol is available for the Sciclone® NGSx workstation

• Functionally validated with Illumina® sequencing platforms

Kit Specs

KIT CONTENTS

• NEXTFLEX® RNA Fragmentation Buffer

• NEXTFLEX® First Strand Synthesis Primer

• NEXTFLEX® First Strand Synthesis Buffer Mix

• NEXTFLEX® Rapid Reverse Transcriptase

• NEXTFLEX® Second Strand Synthesis Mix

• NEXTFLEX® Adenylation Mix

• NEXTFLEX® Ligation Mix

• NEXTFLEX® RNA-Seq Barcode Adapter 1 (0.6 μM)

• NEXTFLEX® Primer Mix (12.5 μM)

• NEXTFLEX® PCR Master Mix

• Nuclease-free Water

• Resuspension Buffer

REQUIRED MATERIALS NOT PROVIDED

• 10 ng – 1 µg total RNA for enrichment by NEXTFLEX® Poly(A) Beads 2.0 or ~ 1 ng – 100 ng isolated mRNA or rRNA-depleted RNA

• DynaMag™-2 Magnet (Life Technologies Cat # 123-21D)

• Commercial kits for rRNA depletion (optional, if not using Poly(A) enrichment)

• 100% Ethanol (stored at room temperature)

• 80% Ethanol (stored at room temperature)

• 2, 10, 20, 200 and 1000 μL pipettes

• RNase-free pipette tips

• Nuclease-free 1.5 mL microcentrifuge tubes

• Thin wall nuclease-free 0.5 mL microcentrifuge tubes

• 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar

• Adhesive PCR Plate Seal (Bio-Rad, Cat # MSB1001)

• Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881)

• Magnetic Stand -96 (Thermo Fisher®, Cat # AM10027) or similar for post PCR cleanup

• Microcentrifuge

• Thermocycler

• Heat block

• Vortex

Selected Publications that Cite the Use of the NEXTFLEX Rapid RNA-Seq Kit:

Boutrin, M.-C., et al. (2015) A putative TetR regulator is involved in Nitric Oxide stress resistance in Porphyromonas gingivalis. Molecular Oral Microbiology. doi: 10.1111/omi.12128.

Páneka, T., et al. (2016) First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group. Molecular Phylogenetics and Evolution. doi:10.1016/j.ympev.2016.01.011.

Park, S. J., et al. (2014) Optimization of crop productivity in tomato using induced mutations in the florigen pathway. Nature Genetics. 46, 1337–1342. doi:10.1038/ng.3131.

Tan, M. H., et al. (2015) First comprehensive multi-tissue transcriptome of Cherax quadricarinatus (Decapoda: Parastacidae) reveals unexpected diversity of endogenous cellulose. Organisms Diversity & Evolution. Pg 1 – 16. Doi: 10.1007/s13127-015-0237-3.