The NVIGEN NERNST-Seq™ assay provides a highly sensitive RNA sequencing sample preparation workflow. The workflow is enabled by our highly sensitive magnetic nanoparticles that can capture RNA and DNA molecules in a high yield and high fidelity manner. The resulting RNAseq libraries provide high quality sequencing data such as high sequencing read quality, high mapping rate to human genome, and more useful data that are mapped to biotypes such as protein coding region and long non-coding RNA region. Ribozyme RNA are removed with negligible presence in the final RNA sequencing libraries.
Figure 1. NVIGEN magnetic nanoparticle capture of single cell RNA samples provided higher RNAseq library yield and better quality of sequencing data, with majority of sequencing reads aligned to protein coding region.
NERNST-Seq™ works with minuscule quantity of RNA samples, for example, sub-single cell RNA content that is composed of only 1-5% of single cell RNA material. RNA samples from as low as 50 fg to 100 ng level have been successfully applied to provide high quality RNA sequencing libraries with the NERNST-Seq™ Assay.
NERNST-Seq™ Assay Workflow includes following steps, in each step, NVIGEN magnetic nanoparticle products have been applied to capture the useful RNA or DNA molecules to provide high quality RNA sequencing sample for high quality NGS data. Below is a table listing the main steps in the NERNST-Seq™ Assay Workflow and the products NVIGEN offer:
|Step and Function
|Products Catalog #
|Add to cart
|NERNST-Seq™-Tissue Fixation Solution
|Tissue Fixation Solution onto Glass Slides for Imaging, Single Cell Analysis, and Intracellular Content Sampling.
for 50 Fixations of Inch Size Tissue Slices
|NERNST-Seq™-mRNA capture kit
|Capture mRNA from total RNA samples to prepare RNA sequencing library.
for 2000 Reactions
|MagVigen™ DNA cleanup and size selection kits
|DNA fragments cleanup and size selection in the RNA sequencing library preparation workflow during reverse transcription-cDNA Amplification-NGS library amplification process. K61001-Easy works for DNA selection size cutoff of 100 to 500 bp; K61001-Extend works for DNA selection size cutoff of larger than 500 bp.
|NERNST-Seq™-Reverse Transcription Enhancer
|To improve the yield of reverse transcription and downstream RNA sequencing libraries.
|NR81005-RT Enhance Buffer
for 2000 Reactions
single cell RNA sequencing library preparation kit
|The kit works with RNA quantity from as low as 50 fg to 100 ng level, suitable for single cell or small quantity of RNA sequencing library sample preparation.
|NR81005-scRNA lib kit
for 96 Libraries
|Complete solution of sample to sequencing result for single cell or minuscule quantity of RNA samples.
- High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality.
Example Experiment Data in NERNST-Seq™-Assay
NERNST-Seq™-mRNA Capture Kit
Figure 2. NERNST-Seq™-mRNA Capture Kit utilize a specially designed biotin-polyT-oligo and MagVigen™ magnetic nanoparticle-streptavidin conjugates. Compared to other on market magnetic beads-streptavidin conjugates, our MagVigen™ magnetic nanoparticle-streptavidin conjugates result in higher yield of RNA sequencing libraries.
Figure 3. Bioanalyzer profile of individual RNA sequencing libraries prepared from 100 pg of breast tissue RNA with and without magnetic nanoparticle mRNA capture. The blue curve shows the RNA sequencing library prepared with the NERNST-Seq™ magnetic nanoparticle mRNA capture step. The red curve shows the RNA sequencing library without magnetic nanoparticle mRNA capture. The magnetic nanoparticle mRNA capture step significantly increased the RNA sequencing library yield. There are also quite a few peaks with DNA fragment sizes from 30-150 bp range depicting the primers, adaptors and their dimers and multimers in the individual RNA sequencing libraries.
Figure 4. Bioanalyzer profile shows clean pooled RNA sequencing library after individual RNA sequencing libraries were pooled together and cleaned up using MagVigen™ Easy DNA select magnetic nanoparticle with 1:1 ratio of RNA sequencing library vs. nanoparticle ratio. The primers, adaptors and their dimers and multimers are removed clean after the beads cleanup step. Such pooled and cleanuped RNA sequencing libraries were loaded onto a sequencer to generate RNAseq data.
High Quality Single Cell RNA Sequencing Data
Figure 5. NERNST-Seq™-assay provided RNA sequencing libraries from single cell or minuscule quantity of RNA samples with high NGS read quality. The Phred Score are mostly better than 30 over the 150 bp size range of the reads, for both read1 and read2. The figure shows the sequencing read quality of RNA sequencing libraries prepared with a variety of conditions with varied NERNST-Seq™-mRNA capture magnetic nanoparticle surface chemistry or capture buffer components, at either 33 pg or 100 pg RNA sample quantity.
NERNST-Seq™ High Quality RNAseq Data with Majority Reads Mapped to Protein Coding and LNC RNA Sequences
Figure 6. NERNST-Seq™-RNA sequencing assays provided high quality alignment rates of RNAseq data to more useful biotypes. Over 70% of RNAseq Data were aligned to protein coding sequences, followed by 10-20% aligned to long noncoding RNA (lncRNA) sequences and the rest of biotypes such as mitochondria or ribozyme RNA reads were negligible.
Top 50 Gene Counts from an RNA Sequencing Sample
Figure 7. Example of the top 50 gene counts from an RNA sequencing sample with the NERNST-Seq™-assay. No mitochondria or ribosome RNAs showing up. These genes can provide better biological insights from single cell or other minuscule quantity of RNA sequencing experiments.