FOR CONSTRUCTION OF MOLECULAR INDEXED™ RNA-SEQ AND ChIP-SEQ LIBRARIES
NEXTFLEX® Illumina® RNA-Seq Library Prep Kit v2 with Molecular Indexes
- Enables high precision measurement of unique RNA-seq or ChIP-seq reads by molecular indexing
- Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads
- 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 or 500 pg ChIP-DNA
- Up to 96 samples can be multiplexed
- Automation-friendly workflow is compatible with liquid handlers
- Functionally validated with Illumina sequencing platforms
For research use only. Not for use in diagnostic procedures.
High Precision Gene Analysis
The patent pending NEXTFLEX qRNA-Seq kit v2 enables high precision gene expression analysis by RNA-Seq and quantitative detection of immunoprecipitated DNA by ChIP-Seq. Developed in conjunction with Cellular Research Inc., this kit generates libraries equivalent to conventional RNA-Seq or ChIP-Seq libraries, but with the added benefit of molecular indexing. Prior to any PCR amplification steps, all cDNA or ChIP-DNA fragment ends are ligated at random to a pair of adapters containing a unique molecular index. Whereas in conventional library preparations unique adapter ligation events onto an identical DNA sequence is indistinguishable from PCR duplicates, molecular indexing allows for differentiation between unique starting molecules and PCR duplication events. Analysis using molecular indexing information provides an absolute, digital measurement of gene expression levels, irrespective of common amplification distortions observed in many NGS experiments.
Cell Free DNA-Seq Multiplexing Options
The NEXTFLEX® qRNA-Seq™ Kit v2 contains two sets of 96 distinct Molecular Index Adapters each of which contains an 8 nucleotide molecular index. In the ligation reaction, these 96 adapters are present in vast molar excess over the concentration of the DNA fragments, and therefore serve as a non-depleting reservoir of molecular indices. Each end of a DNA fragment independently and randomly becomes ligated to a single label from this pool of molecular indices to result in a total of 9,216 (96 x 96) possible combinations across both ends. For every clone sequenced, paired-end reads reveal the chosen label on each end along with the adjoining library sequence. In addition to molecular indexing of individual library inserts, barcoded primers are incorporated into each library to allow for multiplexed sequencing runs.
Using the NEXTFLEX® qRNA-Seq™ Kit v2 for ChIP-Seq
The NEXTFLEX® qRNA-Seq™ Kit v2 can now also be used with ChIP samples. During NGS library preparation, a ChIP sample is ligated to a pair of adapters containing a unique molecular index. Molecular indexing thus allows identical DNA molecules in the starting material to be distinguished from PCR duplicates, allowing accurate quantitative analysis of ChIP-seq data. In addition, molecular indexing is an ideal technology for studying SNPs, as polymerase errors can be easily distinguished from true polymorphisms. Protocols for both DNA and RNA starting material are available.
Using the NEXTFLEX® qRNA-Seq™ v2 Illumina® library prep kit, mRNA or rRNA-depleted RNA is fragmented using a cationic buffer. Fragmented RNA undergoes first and second strand synthesis, followed by end-repair, adenylation, ligation and PCR.
Using the NEXTFLEX® qRNA-Seq™ v2 Illumina® library prep kit, ChIP DNA undergoes end-repair, adenylation, ligation and PCR.
The NEXTFLEX® qRNA-Seq™ Kits v2 contain Illumina® barcoded primers so barcodes do not need to be purchased. The 8 reaction kit includes 4 barcoded primers for multiplexing. Four different versions of the 48 reaction kit are available, each contain 24 unique barcoded primers. Up to 96 unique barcoded primers are available.
- NEXTFLEX® RNA Fragmentation Buffer
- NEXTFLEX® First Strand Synthesis Primer
- NEXTFLEX® First Strand Synthesis Buffer Mix
- NEXTFLEX® Second Strand Synthesis Mix
- NEXTFLEX® End Repair Buffer Mix
- NEXTFLEX® End Repair Enzyme Mix
- NEXTFLEX® Adenylation Mix
- NEXTFLEX® Ligation Mix
- NEXTFLEX® Molecular Index Adapters
- NEXTFLEX® qRNA-Seq™ Universal Forward Primer
- NEXTFLEX® qRNA-Seq™ PCR Master Mix
- NEXTFLEX® qRNA-Seq™ Barcoded Primers
- Nuclease-free Water
- Resuspension Buffer
REQUIRED MATERIALS NOT PROVIDED
- RNA 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
- ChIP DNA input: 500 pg – 100 ng ChIP DNA
- NEXTFLEX® Poly(A) Beads (Bioo Scientific Cat. #512981)
- DynaMag™-2 Magnet (Life Technologies Cat # 123-21D)
- Ribo-Zero™ (Epicentre) / or / RiboMinus™ (Life Technologies) for rRNA depletion
- SuperScript® III Reverse Transcriptase (Life Technologies Cat # 18080-093)
- 100% Ethanol (stored at room temperature)
- 80% Ethanol (freshly prepared)
- 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 (BioRad, Cat # MSB1001)
- Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881)
- Magnetic Stand -96 (Ambion, Cat # AM10027) or similar for post PCR cleanup
- Heat block
- Sequences of NEXTFLEX® qRNA-Seq v2 Barcoded Primers Indexes – Excel / PDF
- Sequences of NEXTFLEX® qRNA-Seq v2 Molecular Indexes A1-A96 – Excel / PDF
qRNAseq STL Script for Accurate Molecular Quantification of mRNA Transcription with Molecular Indexes™
Do I have to perform paired-end sequencing with this kit?
Paired-end sequencing is necessary to take advantage of molecular indexing, but libraries created with this kit are compatible with single-read sequencing.
Is multiplexing possible with this kit?
Yes, multiplexing is possible and there are up to 96 unique barcodes available for multiplexing.
When are the molecular indexes read?
The molecular indexes are read as the first 8 bases of each read.
What sequencing platforms is this kit compatible with?
This kit is compatible with all common Illumina® sequencing platforms including HiSeq®, MiSeq®, and NextSeq® 500 systems.
Is this kit compatible with DNA starting material?
Yes. There is an option for DNA starting material in the manual.
The NEXTFLEX® qRNA-Seq Kit v2 contains enough material to prepare 8 or 48 RNA or ChIP samples for Illumina® compatible paired end sequencing. The shelf life of all reagents is 12 months when stored properly. All components can safely be stored at -20°C.