NEXTflex® Rapid Illumina RNA-Seq Library Prep Kit

Rapid RNA-Seq library prep for sequencing on Illumina platforms

Catalog# Product Name Quantity US List Price
NOVA-5138-01 NEXTflex® Rapid RNA Sequencing Kit 8 rxns $337 Buy Now
NOVA-5138-02 NEXTflex® Rapid RNA Sequencing Kit 48 rxns $1,796 Buy Now
NOVA-512911 NEXTflex® RNA-Seq Barcodes - 6 48 rxns $233 Buy Now
NOVA-512912 NEXTflex® RNA-Seq Barcodes - 12
96 rxns $460 Buy Now
NOVA-512913 NEXTflex® RNA-Seq Barcodes - 24
192 rxns $910 Buy Now
NOVA-512914 NEXTflex® RNA-Seq Barcodes - 48
384 rxns $1,791 Buy Now
NOVA-512915 NEXTflex-96™ RNA-Seq Barcodes - 96
768 rxns
$3,463
Buy Now
NOVA-512979 NEXTflex® Poly(A) Beads
8 rxns
$34
Buy Now
NOVA-512980
NEXTflex® Poly(A) Beads 48 rxns $151 Buy Now
NOVA-512981
NEXTflex® Poly(A) Beads 100 rxns  $302
Buy Now

NEXTflex Rapid Illumina RNA-Seq Library Prep Kit
  • 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 or ~ 1 ng - 100 ng isolated mRNA or rRNA-depleted RNA
  • 96 barcodes are available for multiplexing
  • Automation protocol is available for the Sciclone® NGS Workstation
  • Functionally validated with Illumina® sequencing platforms

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.


Multiplexing up to 96 RNA Libraries for Illumina® Sequencing

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® NGS Workstations is now available. Download the Sciclone NGS and NGSx Workstation Automation Guide for the NEXTflex Rapid RNA-Seq Kit.


Magnetic Beads for mRNA Purification

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


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.


Kit Specs

The NEXTflex Rapid RNA-Seq Illumina® library prep kit contains enough material to prepare 8 or 48 RNA samples for RNA seuencing on an Illumina® platform.. The shelf life of all reagents is 12 months when stored properly. All components can safely be stored at -20°C.

NEXTflex® Rapid RNA-Seq Protocol

 

NEXTflex Rapid RNA-Seq Workflow Illumina Compatible

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 or ~ 1 ng - 100 ng isolated mRNA or rRNA-depleted RNA NEXTflex® Poly(A) beads
DynaMag™-2 Magnet (Life Technologies Cat # 123-21D)
Ribo-Zero™ (Epicentre) or RiboMinus™ (Life Technologies) for rRNA depletion
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

Streamlined Library Construction for Quantitative, Directional, and Standard RNA-Seq

RNA Sequencing (RNA-Seq) is a valuable tool for a broad range of clinical, environmental, and basic research.  Producing high quality RNA-Seq libraries can be challenging for several reasons, including isolation of pure RNA, efficiently converting RNA to cDNA, and loss of material incurred during the series of enzymatic steps and cleanups required for library construction. Here we introduce Bioo Scientific’s family of Illumina compatible NEXTflex® Rapid RNA-Seq kits, all of which include the thermostable NEXTflex™ Rapid Reverse Transcriptase enzyme.  The NEXTflex Rapid RNA-Seq Kits provide affordable and unique technology, some of the shortest RNA-Seq library preparation times of any kits on the market, include all enzymes required for library preparation, and produce the highest quality RNA-Seq library. 

 

Introduction

The NEXTflex™ Rapid RNA-Seq Kits allow the end user to complete library construction in less than 1 day.  In contrast, the previous generation of NEXTflex™ RNA-Seq Kits, as well as the current Illumina TruSeq RNA-Seq Kits, require over 7 hours for completion (Table 1). By combining second strand synthesis and end repair into the same reaction, the need for a separate end repair step and cleanup is eliminated. This improvement is time efficient and reduces loss of material during cleanup.

 

Kit

Time to Completion

Steps Required

Bead Cleanups Required

Reverse Transcriptase Included?

NEXTflex™ Rapid RNA-Seq Kit

5 hours 50 min

6

3

Yes

Illumina TruSeq™ RNA V2

7 hrs 10 min

7

4

No

 

Table 1. Comparison of RNA-Seq library preparation kit protocols and time to completion. Note that times include bead cleanups and account for time required from RNA fragmentation to the final bead cleanup after PCR.

 

Thermostable NEXTflex™ Rapid Reverse Transcriptase for Improved First Strand Synthesis and Library Yield

A critical step in RNA-Seq library construction is the conversion of RNA to cDNA. All NEXTflex Rapid RNA-Seq Kits include NEXTflex™ Rapid Reverse Transcriptase (RT) enzyme, in contrast to our previous RNA-Seq kits as well as to the Illumina TruSeq kit (Table 1). The NEXTflex Rapid RT is a thermostable, RNaseH minus enzyme that functions optimally at 50˚ C, a higher temperature than standard Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptases, which function at 42˚ C.  This elevated temperature allows for reduced secondary structure in RNA templates and therefore increased efficiency of first strand synthesis.  

To examine the efficacy of NEXTflex Rapid RT in library construction, we compared library yields using cDNA produced by different enzymes.  The same pool of Poly (A)+ selected mRNA isolated from murine Ag8 cells was used in first strand synthesis with either NEXTflex Rapid RT or SuperScript® III (Figure 1). Higher library yields were obtained using the NEXTflex Rapid RT coupled with the NEXTflex Rapid RNA-Seq Kit. Similar results were obtained using the NEXTflex™ Rapid Directional and NEXTflex™ Rapid Directional qRNA-Seq™ Kits. These results demonstrate improved library yields as a result of optimized first strand synthesis using NEXTflex Rapid RT.

 

 

 NEXTflex Rapid reverse transcriptase compared with Life Technologies' SuperScript III

Figure 1.  Improved library yield using NEXTflex Rapid RT.  High Sensitivity DNA Bioanalyzer traces of RNA-Seq libraries constructed with NEXTflex Rapid RNA-Seq Kits.  Libraries were constructed using 10 ng of fragmented, Poly (A)+ mRNA converted to cDNA using either NEXTflex Rapid RT (blue) or SuperScript® III (red).

 

High-quality RNA-Seq Data

Further analysis of NEXTflex Rapid RNA-Seq library quality was carried out using Illumina Sequencing, as library yield is only one metric of library quality.  Libraries were prepared with the NEXTflex Rapid RNA-Seq Kit using 10 ng aliquots of a single murine Ag8 cell Poly (A)+ RNA sample, so as to disentangle biological variation from technical variation. Libraries were sequenced on a HiSeq2500 using a 67 bp single end RAPID run.  Resulting reads were trimmed based on a quality score moving window using sickle and mapped to the UCSC mm10 assembly using TopHat 2.0.10. We obtained a total of 31,949,336 reads for Rapid RT libraries and 31,720,922 for Superscript III libraries (Table 2). For transcript representation purposes, only reads mapping to exons, specifically all annotated 5’ UTRs, coding sequences, and 3’ UTRs, were further considered. The number of total reads as well as unique reads mapping to exons were similar between the two enzymes; however, a greater number of transcripts, 12,418 vs. 12,129, were represented in the NEXTflex Rapid RT libraries vs. SuperScript III libraries, respectively. 

 

 Rt Enzyme 

Total Reads 

Reads After Quality Trimming 

Unique Reads Mapping to Exons 

Unique Reads in Exons (%) 

Number Transcripts Represented 

 NEXTflex™ Rapid RT

31,949,336 

31,891,608 

17,411,368 

54.60 

12,418 

 Superscript® III RT

 31,720,922

31,663,364 

17,487,086 

55.23 

12,129 

 

Table 2. Read counts in NEXTflex™ Rapid RNA-Seq libraries and unique reads after mapping to exons.

 

A further analysis of mapped reads was performed. Read quality before and after mapping was visualized using FastQC and GC content as a function of reads examined (Figure 2). Read quality is excellent in the total library and is slightly higher in the remaining set of trimmed, mapped reads, which is expected given a quality-aware trimming step was carried out. Furthermore, GC content is very similar to the theoretical distribution, indicating accuracy in transcript representation. Visual inspection of several genes demonstrates read coverage across all exons (Figure 3A and B). Furthermore, a metagene analysis of read coverage across all genes divided into 100 bin segments, demonstrates even read coverage across the 5’-, gene body, and 3’-ends of transcripts (Figure 3C).  

 

Rapid RNA-Seq Fast QC data

Figure 2. Metrics of NEXTflex Rapid RNA-Seq data determined by FastQC. Quality score plots for (top) and mean GC content (bottom) for (A) all reads or (B) reads mapping to exons. Plots shown correspond to NEXTflex Rapid RNA-Seq libraries constructed using NEXTflex Rapid RT. 

 

 

Rapid RNA-Seq read coverage compared to Life Technologies' Superscript III

Figure 3. Read coverage across gene bodies.  Mapped reads at the (A) Trp53 and (B) Eif5b loci scaled to read density as indicated. (C) Metagene plot of read density across all annotated loci. All gene bodies and mapped read densities are scaled to 100 bin segments; mean read density is shown in reads per kilobase per million mapped reads (RPKM; solid line) +/- standard error across replicates (faded bands). Shown is read signal corresponding to libraries made with either NEXTflex Rapid RT (red) or SuperScript III (blue).  

 

Finally, we examined library consistency across protocols. We compared RNA-Seq read counts in exons from libraries constructed using the NEXTflex Rapid RNA-Seq Kit to longer traditional protocols. Indeed, libraries constructed using the two methods were highly similar (r = 0.9976; Figure 4). These data demonstrate that the NEXTflex Rapid RNA-Seq protocol provides significantly faster library construction with enhanced reverse transcriptase performance.  

graph showing rapid RNA-Seq as accurate as standard

 

Figure 4.  Correlation between Rapid and Standard RNA-Seq data. Pearson’s correlation between the average log2 of normalized counts per million (CPM) of all genes across all replicates of Rapid RNA-Seq vs. Standard RNA-Seq, r = 0.9976.

 

Conclusions

The present study demonstrates the utility of the NEXTflex Rapid RNA-Seq Kits as a method to improve speed of library preparation without any compromise in library quality. All NEXTflex Rapid RNA-Seq Kits include NEXTflex Rapid RT, a high performance thermostable enzyme, thus providing all components required for library construction within a single kit. NEXTflex Rapid RNA-Seq Kits produced high-quality sequencing data with improved transcript representation. In addition, the increased library yield using NEXTflex™ Rapid RT is an important improvement for users with low-input amounts.  For researchers wishing to multiplex libraries, Bioo Scientific continues to offer our full range of up to 96 NEXTflex™ RNA-Seq adapter barcodes. The NEXTflex Rapid RNA-Seq, Rapid Directional RNA-Seq, and Rapid Directional qRNA-Seq™ Kits provide a streamlined workflow for users to create high-quality RNA-Seq libraries with less hands-on time.

 

Relative Levels of ERCC Control RNAs in RNA-Seq Libraries 

RNA-Seq library w/ ERCC controls

 

Made from NEXTflex Poly(A) Bead Selected and Total RNA Correlation between expected and observed values for percentage of reads mapping to 94 ERCC control RNAs in RNA-Seq library made using 20 µL of NEXTflex Poly(A) Beads.  

 

Relative Levels of Different Categories of Endogenous Transcripts Detected in RNA-Seq Libraries made from NEXTflex Poly(A) Bead Selected and Total RNA 

 

RNA-Seq library prep endogenous transcripts

 

Percentage of reads mapping to rRNA regions and regions of interest with and without Poly(A) bead selection.